Cd38 modulating antibody agents

ABSTRACT

The present disclosure provides antibody sequences found in antibodies that bind to human CD38. In particular, the present disclosure provides sequences of anti-human CD38 antibodies. Antibodies and antigen-binding portions thereof including such sequences present features compatible with pharmaceutical manufacturing and development can be provided as fully human antibodies (e.g., fully human monoclonal antibodies or antigen-binding fragments) that can be useful for medical methods and compositions, in particular for treating cancer.

BACKGROUND

CD38 is a type II membrane receptor glycoprotein having enzymaticactivities, in particular as an important ADP-ribosyl cyclase thatproduces cyclic adenosine diphosphate ribose (cADPR) from nicotinamideadenine dinucleotide. Different extracellular stimuli can induce cADPRproduction. cADPR is important to the mobilization of intracellularcalcium stock that is involved in many cell functions such as cellproliferation, differentiation, adhesion, and signal transduction. CD38was initially identified as a leukocyte activation marker but plays dualroles as receptor and ectoenzyme, endowed with cell signalling and cellhomeostasis activities. CD38 has been linked to various human diseases,including malignancies such as chronic lymphocytic leukemia, myeloma andovarian carcinoma (Quarona V, et al., 2013; Wei W, et al., 2014).

CD38 is found on the surface of many cell types that are involved inimmunological responses (in short referred to as immune cells),including effector cells such as T and B lymphocytes and NK cells, butalso immune suppressive cells such as regulatory T and B cells, myeloidderived suppressive cells (MDSCs) or tumour associated macrophages(Chevrier S et al. 2017). For instance, in lung cancer patients,anti-PD-1 treatment induced proliferation of PD-1 expressing T cellsthat expressed high levels of CD38 (Kamphorst A O et al., 2017). Theimportance of cADPR- and CD38-mediated Ca²+ signalling for biologicalactivity of immune cells, in particular for the modulation of the immuneresponse in physiological and pathological conditions, has beendescribed in the literature (Morandi F et al., 2015; Rah S Y et al.,2015).

CD38 is highly expressed by cancer cells in multiple myeloma patients atall stages of disease and in CLL patients with a poor prognosis. VariousCD38-targeting therapies are developed by generating compounds that actmainly as CD38 antagonists or inhibitors (de Weers M et al., 2011; vande Donk N W et al., 2016; Horenstein A L et al., 2017). Anti-CD38monoclonal antibodies acting as CD38 agonists (such as the one namedIB4) have also been characterized as inducing the mobilization ofcalcium ions, CD38 shedding, NK cell-mediated cytotoxicity, cytokinesecretion (in particular Interleukin 6 and Interferon gamma), andproliferation of human T lymphocytes, among other activities, and weremodified to generate immunotoxins (Malavasi F et al., 2008;Hara-Yokoyama M et al., 2008; Frasca L et al, 2006; Karakasheva T etal., 2015). Such a positive effect on immune cells may be related toinduction of Ca2+ mobilization, inhibition of CD38 enzymatic activity,and/or activation of intra-cellular signaling pathways.

Monoclonal antibodies were developed for targeted, direct killing ofCD38-expressing tumor cells and have shown promising results in theclinic. However, the activity of such anti-CD38 antibodies may berestricted to tumors in which CD38 is highly expressed on surface ofcancer cells. In solid tumors, the expression of CD38 is generally loweror absent on the tumor cells and may be associated with the tumorinfiltrating immune cells, both effector and suppressive. Therefore,there is still a need for anti-CD38 antibodies presenting activitiesthat result from the combination of different components, such asCD38-specific agonistic or modulating properties together with targetedcell killing or activation, and compatibility with pharmaceuticaldevelopment, and that can be exploited for treating cancer, inparticular for treating solid cancers.

SUMMARY

In some embodiments, the present invention provides new CD38 ModulatingAntibody Agents. In some embodiments, provided CD38 Modulating AntibodyAgents are antibodies or antigen-binding fragments that specificallybind to CD38, and particularly to human CD38, in many embodiments to asite in the human CD38 extracellular domain.

In some embodiments, provided antibodies or antigen binding fragmentsmodulate one or more features of CD38. That is, in some embodiments,level and/or activity of CD38, and/or one or more downstream effectsthereof, is detectably altered when a provided antibody is present ascompared with when it is absent. Alternatively or additionally, in someembodiments, level and/or activity of CD38, and/or one or moredownstream effects thereof, when a provided antibody is present, iscomparable to or greater than that observed under comparable conditionswhen a reference CD38 Modulating Antibody Agent (e.g., a referenceanti-CD-38 antibody, such as IB-4, with a known desirable attribute;e.g., a known ability to agonize one or more features of CD38).

In many embodiments, one or more features of CD38 is enhanced when aprovided CD38 Modulating Antibody Agent (e.g., anti-CD38 antibody orantigen-binding fragment thereof) is present. For example, in someembodiments, presence of a provided CD38 Modulating Antibody Agent(e.g., anti-CD38 antibody or antigen-binding fragment thereof)correlates with increased immune cell activation, and/or proliferation.Thus, provided CD38 Modulating Antibody Agents are often referred toherein as “agonists”. Those skilled in the art, however, will appreciatethat teachings of the present disclosure are not limited by particularmechanism of action of provided antibodies or antigen-binding fragmentsthereof. Relevant structural and/or functional features of providedantibodies are described herein and speak for themselves.

In some embodiments, provided CD38 Modulating Antibody Agents (e.g.,CD38 antibodies or antigen-binding fragments) may be characterized, forexample, by effects on certain immune effector cells (e.g., NK cellsand/or T cells). Alternatively or additionally, in some embodiments,provided CD38 Modulating Antibody Agents (e.g., CD38 antibodies orantigen-binding fragments) may be characterized, for example, by effectson immune suppressive cells. For example, in some embodiments, providedCD38 Modulating Antibody Agents display activating properties withrespect to immune effector cells such as NK cells and T cells andcytotoxic properties towards CD38 high expressing cells such as immunesuppressive cells. Alternatively or additionally, in some embodiments,provided CD38 Modulating Antibody Agents are characterized by one ormore features that are associated with binding to a specific epitope inhuman CD38 extracellular domain and/or that render them particularlyamenable to pharmaceutical use and/or manufacturing.

Provided technologies, including provided CD38 Modulating AntibodyAgents (e.g., provided antibodies or antigen-binding fragments thereof(or variants of the same)), compositions including them, and/or uses forthem, are useful in medicine. In some embodiments, such providedtechnologies are useful in cancer therapy and/or prophylaxis.

In some embodiments, provided CD38 Modulating Antibody Agents areexemplified by the antibodies having the sequence of aCD38-b-329, andmore in general antibodies or agents that are or comprise one or moreantigen-binding fragments or portions thereof, for example that comprisethe aCD38-b-329-HCDR3 amino acid sequence (SEQ ID NO: 3) as variableheavy chain complementarity determining region 3, and/or, in someembodiments, comprise one or both of the aCD38-b-329-HCDR1 (SEQ IDNO: 1) and HCDR2 (SEQ ID NO: 2) sequences, and/or that compete withaCD38-b-329 for binding human CD38 extracellular domain. In someembodiments, provided antibodies or antigen-binding fragments thereofbind to human CD38 with a Kd of in the 10⁻⁸ M range, or below (in the10⁻⁹ M range). In some embodiments the Kd is from 10⁻⁸ to 10⁻⁹. The Kdto evaluate the binding affinity of the antibodies or antigen bindingfragments thereof can be obtained by standard methodologies includingsurface plasmon resonance (SPR) such as Biacore analysis or analysisusing Forte Bio Octet Systems.

In some embodiments, provided CD38 Modulating Antibody Agents (e.g.,provided antibodies or antigen-binding fragments thereof) bind to anepitope on human CD38 that is bound by aCD38-b-329. In some embodiments,such provided CD38 Modulating Antibody Agents may bind to human CD38extracellular domain. In some embodiments, provided CD38 ModulatingAntibody Agents may bind to an epitope of CD38 (e.g., when assessedusing one or more assays as described herein or otherwise known in theart), in particular the one identified as aCD38-b-ep. In someembodiments, provided antibodies or antigen-binding fragments thereofmay bind to human and Cynomolgus Monkey CD38 (e.g., to an extracellularepitope on human and Cynomolgus Monkey CD38) with Kd value in the 10⁻⁸ Mrange.

In some embodiments, provided antibodies or antigen-binding fragmentsthereof bind to a mutant human CD38 (as compared to non-mutant humanCD38 (SEQ ID NO: 9)), wherein in the mutant human CD38, the serineresidue in position 274 has been substituted with a phenylalanine.

In some embodiments, provided antibodies or antigen-binding fragmentsthereof bind to a mutant human CD38 (as compared to non-mutant humanCD38 (SEQ ID NO: 9)), wherein in the mutant human CD38, the aspartateresidue in position 202 has been substituted with a glycine residue.

In some embodiments, provided antibodies or antigen-binding fragmentsthereof bind to a mutant human CD38 (as compared to non-mutant humanCD38 (SEQ ID NO: 9)), wherein in the mutant human CD38, the serineresidue in position 274 has been substituted with a phenylalanine andthe aspartate residue in position 202 has been substituted with aglycine residue.

Among other things, the present disclosure provides a procedure (FIG. 1)that can be utilized to identify and/or characterize particularly usefulCD38 Modulating Antibody Agents (e.g., anti-CD38 antibodies orantigen-binding fragments thereof) as described herein (e.g., anti-CD38antibodies or antigen-binding fragments thereof characterized by certainstructural and/or functional features, such as specific binding to humanCD38 (e.g., to an extracellular epitope thereof), inclusion of one ormore CDR sequence elements as described herein (and particularlyinclusion of an HCDR3 sequence element, optionally in combination withHCDR1 and/or HCDR2 elements), cell activating activity as describedherein, cytotoxic activity as described herein (e.g., with respect toimmune regulatory cells with relatively high levels of CD38 on theirsurfaces), and combinations thereof). In some embodiments, particularlyuseful anti-CD38 antibodies as described herein are characterized by aplurality of such features. In some embodiments, one or more antibodiesdescribed herein may be characterized as a CD38 Modulating AntibodyAgent.

Thus, as exemplified herein, certain antibodies and/or antigen-bindingfragments comprising aCD38-b-329 sequences (in particularaCD38-b-329-HCDR3 (SEQ ID NO: 3) and/or aCD38-b-329-LCDR3 (SEQ ID NO:7)) are characterized by such desirable structural and/or functionalfeatures; such antibodies and/or antigen-binding fragments thereof maybe referred to herein as CD38 Modulating Antibody Agents. Additionally,in accordance with the present disclosure, antibodies andantigen-binding fragments thereof compete with aCD38-b-329 may beparticularly useful antibodies; such antibodies and/or antigen-bindingfragments thereof may also be referred to herein as CD38 ModulatingAntibody Agents.

Antibodies (and/or antigen-binding fragments thereof) described hereinmay be particularly useful in medicine (e.g., in therapy and/or inprophylaxis, for example in the treatment of cancer), and/or for usewith respect to methods that require or involve targeting an epitopesuch as the one identified as aCD38-b-ep within human CD38 extracellulardomain. Provided antibodies or antigen-binding fragments thereof may beprepared as presenting the most appropriate isotype, in particular humanisotype from the group consisting of IgG1, IgG2, IgG3, and IgG4 isotypeantibodies, more particularly human IgG1.

In one aspect, the present invention provides aCD38-b-329-HCDR3 aminoacid sequence (SEQ ID NO: 3) and polypeptides that include it, such as,for example, antibodies or antigen-binding fragments comprising theaCD38-b-329-HCDR3 amino acid sequence (SEQ ID NO: 3) as variable heavychain complementarity determining region 3. In some embodiments, suchantibody or antigen-binding fragment may be further characterized bycomprising further aCD38-b-329 amino acid sequence elements such as:

-   a) aCD38-b-329-HCDR1 amino acid sequence (SEQ ID NO: 1) as variable    heavy chain complementarity determining region 1; and/or-   b) aCD38-b-329-HCDR2 amino acid sequence (SEQ ID NO: 2) as variable    heavy chain complementarity determining region 2.

In some embodiments, provided antibodies or antigen-binding fragmentsthereof may comprise variable heavy chain complementarity determiningregions defined above (i.e. aCD38-b-329 amino acid sequence elements)further in the correct order, specifically separated by antibody framesequences, such as the one included in aCD38-b-329-HCDR123 amino acidsequence (SEQ ID NO: 4), in particular for exerting correctly theirbinding and functional properties. For example, in some embodiments, aprovided antibody or antigen-binding fragment said thereof can compriseaCD38-b-329-HCDR123 amino acid sequence (SEQ ID NO: 4, or the HCDR1,HCDR2 and HCDR3 sequences thereof) and, optionally:

-   a) aCD38-b-329-LCDR1 amino acid sequence (SEQ ID NO: 5) as variable    light chain complementarity determining region 1;-   b) aCD38-b-329-LCDR2 amino acid sequence (SEQ ID NO: 6) as variable    light chain complementarity determining region 2; and-   c) aCD38-b-329-LCDR3 amino acid sequence (SEQ ID NO: 7) as variable    light chain complementarity determining region 3.

Thus, in some embodiments, the present invention provides an isolatedantibody or antigen-binding fragments thereof comprising a variableheavy chain comprising aCD38-b-329-HCDR123 amino acid sequence (SEQ IDNO: 4). Preferably, such isolated antibody or antigen-binding fragmentsthereof further comprises a variable light chain comprisingaCD38-b-329-LCDR123 amino acid sequence (SEQ ID NO: 8), as described inthe Examples.

In some embodiments the variable heavy chain sequence of aCD38-b-329comprises the sequence:

(SEQ ID NO: 12) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSDYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQYSSGWYAYPFDMWGQGTMVTVSSand the variable light chain sequence of aCD38-b-329 comprises thesequence:

(SEQ ID NO: 15) EIVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQDGAVFTFGG GTKVEIK.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising the sequence of aCD38-b-329-HCDR3 as anHCDR3 and comprising an LCDR3 having a sequence selected from the groupconsisting of aCD38-b-329-LCDR3, aCD38-b-329-m6-LCDR3 andaCD38-b-329-m7-LCDR3.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising the sequence of aCD38-b-329-HCDR1 as anHCDR1, the sequence of aCD38-b-329-HCDR2 as an HCDR2, the sequenceaCD38-b-329-HCDR3 as an HCDR3, the sequence of aCD38-b-329-LCDR1 as anLCDR1, the sequence of aCD38-b-329-LCDR2 as an LCDR2 and comprising anLCDR3 having a sequence selected from the group consisting ofaCD38-b-329-LCDR3, aCD38-b-329-m6-LCDR3 and aCD38-b-329-m7-LCDR3.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising the sequence of aCD38-b-329-HCDR123 as avariable heavy chain region and comprising a variable light chain regionhaving a sequence selected from the group consisting ofaCD38-b-329-LCDR123, aCD38-b-329-m6-LCDR123 and aCD38-b-329-m7-LCDR123.

The present invention also provides an antibody or antigen-bindingfragment thereof comprising the sequence of aCD38-b-329-VH as a variableheavy chain region and comprising a variable light chain region having asequence selected from the group consisting of aCD38-b-329-VL,aCD38-b-329-m6-VL and aCD38-b-329-m7-VL.

The present invention also provides variant antibodies and antigenbinding fragments thereof that have certain % identities relative to areference sequence, such as a CDR sequence or a heavy or light chainvariable sequence of aCD38-b-329. Such antibodies and antigen bindingfragments thereof may also be referred to herein as CD38 ModulatingAntibody Agents.

For example, in some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising an amino acid sequence having at least 90% sequence identityto SEQ ID NO: 12. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising an amino acid sequence having at least 95% sequence identityto SEQ ID NO: 12. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising an amino acid sequence having at least 98% sequence identityto SEQ ID NO: 12. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 15. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 95% sequence identity to SEQ ID NO: 15. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 98% sequence identity to SEQ ID NO: 15. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising the aminoacid sequence of SEQ ID NO: 15.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 12 anda variable light chain sequence comprising an amino acid sequence havingat least 90% sequence identity to SEQ ID NO: 15. In some embodiments theanti-CD38 antibody or antigen binding fragment thereof comprises avariable heavy chain sequence comprising an amino acid sequence havingat least 95% sequence identity to SEQ ID NO: 12 and a variable lightchain sequence comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 15. In some embodiments the anti-CD38antibody or antigen binding fragment thereof comprises a variable heavychain sequence comprising an amino acid sequence having at least 98%sequence identity to SEQ ID NO: 12 and a variable light chain sequencecomprising an amino acid sequence having at least 98% sequence identityto SEQ ID NO: 15. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising the amino acid sequence of SEQ ID NO: 12 and a variable lightchain sequence comprising the amino acid sequence of SEQ ID NO: 15.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 4. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 95% sequence identity to SEQ ID NO: 4. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 98% sequence identity to SEQ ID NO: 4. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising the aminoacid sequence of SEQ ID NO: 4.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 8. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 95% sequence identity to SEQ ID NO: 8. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising an aminoacid sequence having at least 98% sequence identity to SEQ ID NO: 8. Insome embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable light chain sequence comprising the aminoacid sequence of SEQ ID NO: 8.

In some embodiments the anti-CD38 antibody or antigen binding fragmentthereof comprises a variable heavy chain sequence comprising an aminoacid sequence having at least 90% sequence identity to SEQ ID NO: 4 anda variable light chain sequence comprising an amino acid sequence havingat least 90% sequence identity to SEQ ID NO: 8. In some embodiments theanti-CD38 antibody or antigen binding fragment thereof comprises avariable heavy chain sequence comprising an amino acid sequence havingat least 95% sequence identity to SEQ ID NO: 4 and a variable lightchain sequence comprising an amino acid sequence having at least 95%sequence identity to SEQ ID NO: 8. In some embodiments the anti-CD38antibody or antigen binding fragment thereof comprises a variable heavychain sequence comprising an amino acid sequence having at least 98%sequence identity to SEQ ID NO: 4 and a variable light chain sequencecomprising an amino acid sequence having at least 98% sequence identityto SEQ ID NO: 8. In some embodiments the anti-CD38 antibody or antigenbinding fragment thereof comprises a variable heavy chain sequencecomprising the amino acid sequence of SEQ ID NO: 4 and a variable lightchain sequence comprising the amino acid sequence of SEQ ID NO: 8.

Such variant antibodies and antigen binding fragments thereof may retainor exhibit the same (or substantially the same) functional andpharmacological properties as described for the antibodies and antigenbinding fragments thereof having the heavy and light chain variablesequences disclosed herein for aCD38-b-329.

Moreover, aCD38-b-329 amino acid sequences also refer to antibodysequences that are defined by the number of substitution with respect tothe aCD38-b-329 amino acid sequence elements defined above. For example,such sequence may comprise, as variable heavy chain complementaritydetermining region 3 (HCDR3) a sequence containing up to 1, 2, 3, 4, 5,6, 7, 8, 9 or 10, amino acid substitutions within aCD38-b-329-HCDR3 (SEQID NO: 3). In a further embodiment, aCD38-b-329 amino acid sequencesalso refer to antibody sequences comprising, as variable heavy chaincomplementarity determining regions 1, 2 and 3 (HCDR1, HCDR2, and HCDR3)a sequence containing up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, amino acidsubstitutions within aCD38-b-329-HCDR1, aCD38-b-329-HCDR2, andaCD38-b-329-HCDR3, and more preferably a sequence containing up to 1, 2,3, 4, 5, 6, 7, 8, 9 or 10, amino acid substitutions withinaCD38-b-329-HCDR123 (SEQ ID NO: 4), or within SEQ ID NO: 12. In someembodiments aCD38-b-329 amino acid sequences also refer to antibodysequences comprising as a variable heavy chain sequence a sequencecontaining up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, amino acidsubstitutions within the framework regions of the variable heavy chainsequence. The antibodies presenting such aCD38-b-329 amino acid sequenceelements and such substitutions can still present the binding and/orfunctional properties of aCD38-b-329, and of a CD38 Modulating AntibodyAgents in general.

Such aCD38-b-329 amino acid sequences may also comprise, as variablelight chain complementarity determining region 3 (LCDR3) a sequencecontaining up to 1, 2, 3 or 4, amino acid substitutions withinaCD38-b-329-LCDR3 (SEQ ID NO: 7). In a further embodiment, aCD38-b-329amino acid sequences also refer to antibody sequences comprising, asvariable light chain complementarity determining regions 1, 2 and 3(LCDR1, LCDR2, and LCDR3) a sequence containing up to 1, 2, 3, 4, 5, 6,7, 8, 9 or 10, amino acid substitutions within aCD38-b-329-LCDR1,aCD38-b-329-LCDR2, and aCD38-b-329-LCDR3, and more preferably a sequencecontaining up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, amino acidsubstitutions within aCD38-b-329-LCDR123 (SEQ ID NO: 8), or within SEQID NO: 15. In some embodiments aCD38-b-329 amino acid sequences alsorefer to antibody sequences comprising as a variable light chainsequence a sequence containing up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,amino acid substitutions within the framework regions of the variablelight chain sequence. The antibodies presenting such aCD38-b-329 aminoacid sequence elements and such substitutions can still present thebinding and/or functional properties of aCD38-b-329, and of a CD38Modulating Antibody Agent in general.

Accordingly, in one embodiment, the present invention provides ananti-CD38 Antibody Agent (i.e. an antibody or antigen-binding fragmentthereof and variants thereof as described herein, such as variantsmutated to remove the DG motif) comprising:

-   -   a. the variable heavy chain region sequence of aCD38-b-329 (or a        variant thereof, such as an affinity matured variant thereof) or        a variable heavy chain region sequence having up to 5 amino acid        substitutions compared to the variable heavy chain region        sequence of aCD38-b-329 (or a variant thereof, such as an        affinity matured variant thereof); and/or    -   b. the variable light chain region sequence of aCD38-b-329 (or a        variant thereof, such as an affinity matured variant thereof) or        a variable light chain region sequence having up to 5 amino acid        substitutions compared to the variable light chain region        sequence of aCD38-b-329 (or a variant thereof, such as an        affinity matured variant thereof).

The aCD38-b-329 heavy chains that may incorporate amino acidsubstitutions include SED ID NOs 4, and 12. The aCD38-b-329 light chainsthat may incorporate amino acid substitutions include SED ID NOs 8, 13,14, 15, 16 and 17.

The amino acid substitutions preferably do not adversely effect, or notsubstantially adversely effect, the functional properties of theantibodies. The substitutions may therefore be considered conservativeamino acid substitutions. Preferably, when amino acid substitutions dooccur, they occur in a ratio of 1:1, such that the total length of theheavy and/or light chain variable region does not change.

The invention also provides antibodies or antigen-binding fragmentsthereof, wherein the DG motif in the light or heavy chains of theantibodies may be altered, for example to reduce susceptibility toaspartate isomerization and/or wherein any methionine in the light orheavy chains of the antibodies may be altered, for example to reducemethionine oxidation. For example, a DG motif may be altered tosubstitute one or both of the amino acids in the motif with a differentamino acid. For example, such motifs may be mutated to EG, DQ or DA. Amethionine residue may be altered to replaced it with a different aminoacid, for example leucine or phenylalanine.

Accordingly, in some embodiments, the antibodies or fragments thereofprovided herein can be mutated to remove or modify DG motifs, inparticular DG motifs appearing in the CDR regions, as is standard in theart to reduce susceptibility to aspartate isomerisation. Such antibodiesthat have been modified in this may way need to undergo furthermodification (for example affinity maturation) before arriving at afinal sequence.

In one embodiment of the invention, there is provided a variant antibodyhaving CDR1, CDR2 and CDR3 sequences of an antibody as disclosed herein(for example, the CDR1, CDR2 and CDR3 sequences of aCD38-b-329), or thevariable heavy and variable light chain of any antibody as disclosedherein (for example the variable heavy and variable light chain ofaCD38-b-329), but differing from the specified sequence in that at leastone DG motif in the CDRs (if present) has been changed to a differentmotif. The disclosed variants may be used and formulated as describedfor aCD38-b-329.

For example, aCD38-b-329 contains a DG motif in its LCDR3 sequence. Insome embodiments, the aspartate of the DG motif may be changed to adifferent amino acid and/or the glycine of the DG motif may be changedto a different amino acid. In such embodiments, the anti-CD38 antibodyor antigen-binding fragment thereof may be, or may be derived from, forexample, aCD38-b-329. In some of the embodiments the variant antibodiesor antigen-binding fragments thereof have a VL CDR3 sequence as providedin Table 4 (labelled aCD38-b-329-m6, and aCD38-b-329-m7). For example, avariant LCDR3 sequence (for example a aCD38-b-329-m6 variant LCDR3sequence, or aCD38-b-329-m6 variant LCDR3 sequence) can be incorporatedinto an antibody that comprises the LCDR1 and/or LCDR2 sequences ofaCD38-b-329. In one embodiment, a variant LCDR3 sequence (for examplethe aCD38-b-329-m6 variant LCDR3 sequence, or aCD38-b-329-m7 variantLCDR3 sequence) can be incorporated into an antibody that comprises theLCDR1, LCDR2, HCDR1, HCDR2 and HCDR3 sequences of aCD38-b-329. In someembodiments, the variant antibody or antibody binding fragment thereofmay comprise the variable heavy and variable light chain sequences ofaCD38-b-329, but with the LCDR3 sequence mutated to remove the DG motif(for example aCD38-b-329-m6-LCDR3, or aCD38-b-329-m6-LCDR3 may bepresent as a LCDR3 instead). The variant anti-CD38 antibodies providefurther antibodies having any, and possibly all, binding and functionalproperties of the parental aCD38-b-329. The disclosed variants may beused and formulated as described for aCD38-b-329.

Accordingly, the variant antibody aCD38-b-329-m6 may be characterised ascomprising a heavy chain variable region comprising the sequence of:

(SEQ ID NO: 12) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSDYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQYSSGWYAYPFDMWGQGTMVTVSSand a variant light chain comprising the sequence:

(SEQ ID NO: 13) EIVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQDEAVFTFGG GTKVEIK

The variant antibody aCD38-b-329-m7 may be characterised as comprising aheavy chain variable region comprising the sequence of:

(SEQ ID NO: 12) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSDYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQYSSGWYAYPFDPDMWGQGTMVTVSSand a variant light chain comprising the sequence:

(SEQ ID NO: 14) EIVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQDSAVFTFG GGTKVEIK

The invention also provides affinity matured antibodies, for example anaffinity matured variant derived from any of the antibodies disclosedherein. In one embodiment, the affinity matured antibodies are affinitymatured antibodies having an altered DG motif and/or NG motif and/oraltered to remove or mutate any methionine residues. The disclosedaffinity matured variants may be used and formulated as described foraCD38-b-329.

In some embodiments the invention provides a method of preparing ananti-CD38 antibody comprising providing an antibody as herein described(e.g., aCD38-b-329 or an antigen binding fragment or variant thereof),and subjecting the antibody to affinity maturation, wherein the antibodyproduced binds to CD38 with greater affinity than the parental antibody.Preferably the produced antibody binds to CD38 with at least 20%, atleast 30%, at least 40%, more preferably at least 50% greater affinitythan the parental antibody binds to CD38, for example as measured by theKd. Methods for measuring affinity are known in the art and described inthe Examples below. The affinity matured antibodies produced by suchmethods can be formulated and used as described herein for the otheranti-CD38 Antibody Agents.

Affinity maturation may be carried out according to any suitable methodknown to the skilled person. For example, in vitro antibody displaysystems are widely used for the generation of specific antibodies withhigh affinity. In these systems, the phenotype (i.e., the antibodyfragment) is coupled to the genotype (i.e., the antibody gene) allowingthe direct determination of the sequence of the antibody. Severalsystems have been developed to achieve display of antibody repertoiresto allow subsequent selection of binders and by increasing thestringency of selection allows for the selection of higher and higheraffinity variants. The antibody fragments can be expressed in yeast,ribosomes, phage display particles or by direct coupling to DNA.

Current antibody affinity maturation methods belong to two mutagenesiscategories: stochastic and non-stochastic. Error-prone polymerase chainreaction (PCR), mutator bacterial strains, and saturation mutagenesisare typical examples of stochastic mutagenesis methods. Non-stochastictechniques often use alanine-scanning or site-directed mutagenesis togenerate limited collections of specific variants. In addition,shuffling approaches to obtain shuffled variants of the parent antibodycan also be used to improve antibodies affinity further.

Accordingly, in one embodiment of the invention, the method of affinitymaturation is selected from the group consisting of stochasticmutagenesis (for example error-prone polymerase chain reaction (PCR),mutator bacterial strains, or saturation mutagenesis), non-stochasticmutagenesis (for example alanine-scanning or site-directed mutagenesis),shuffling (for example DNA shuffling, chain shuffling or CDR shuffling)and the use of the CRISPR-Cas9 system to introduce modifications.

Affinity maturation methods are described in, for example, Rajpal etal., Proc Natl Acad Sci USA, 2005, 102(24):8466-71, Steinwand et al.,MAbs, 2014, 6(1):204-18, as well as in Handbook of TherapeuticAntibodies, Wiley, 2014, Chapter 6, Antibody Affinity (pages 115-140).

In some embodiments there is provided a method of preparing apharmaceutical composition comprising providing an antibody preparedaccording to a method above, (i.e. for producing an antibody by affinitymaturation) and co-formulating the antibody with at least one or morepharmaceutically acceptable excipients. The antibody used in thepreparation of the pharmaceutical composition can be an affinity maturedvariant of aCD38-b-329. The pharmaceutical compositions produced by suchmethods can be used in the methods of treatment of the present inventionas described herein for the other anti-CD38 Antibody Agents.

Provided antibodies and/or antigen-binding fragments thereof asdescribed herein (e.g., a CD38 Modulating Antibody Agent that mayinclude one or more aCD38-b-329 amino acid sequence elements suchaCD38-b-329-HCDR3 or aCD38-b-329-HCDR123, and/or that may compete withaCD38-b-329 for binding to human CD38 and non-human primate CD38 forexample Cynomolgus monkey CD38, etc.) may be provided in any of avariety of formats. For example, in some embodiments an appropriateformat may be or comprise a monoclonal antibody, a domain antibody, asingle chain antibody, a Fab fragment, a F(ab′)2 fragment, a singlechain variable fragment (scFv), a scFv-Fc fragment, a single chainantibody (scAb), an aptamer, or a nanobody. In some embodiments, anantibody or antigen-binding fragment thereof (and particularly amonoclonal antibody), may be a rabbit, mouse, chimeric, humanized orfully human antibody or antigen-binding fragment thereof. In someembodiments, a provided antibody or antigen-binding fragment thereof maybe of an IgG, IgA, IgE, or IgM isotype (preferably human ones), as itcan be most appropriate for a given use. In some embodiments, a providedantibody or antigen- binding fragment thereof is an IgG isotype, moreparticularly an IgG1, IgG2, IgG3, or IgG4 isotype (preferably humanIgG1). In some embodiments, a provided antibody or antigen-bindingfragment thereof (e.g., is provided as part of a multi-specific bindingagent such as, for example, when it is desirable to associate furtherbinding and/or functional moieties to a CD38 Modulating Antibody agentsuch as a aCD38-b-329 amino acid sequence, the isolated antibody orantigen-binding can be comprised in a bispecific antibody, amultispecific antibody, or other multi-specific format that may beavailable in the art.

In some embodiments, a provided CD38 Modulating Antibody Agent comprisesa CD38-binding entity (e.g., an anti-CD38 antibody or antigen-bindingfragment thereof) and a conjugated payload such as a therapeutic ordiagnostic agent. In many such embodiments, the agent is consideredand/or referred to as an “immunoconjugate”. Examples of technologies andcompounds that can be used for generating specific immunoconjugates suchas antibody-drug are disclosed in the literature (Beck A et al., 2017)and described as applicable to several known anti-CD38 antibodies(WO2016166304).

In some embodiments, the present invention provides aCD38-b-329 aminoacid sequences that identify provided antibodies or antigen-bindingfragments thereof. In some embodiments, such sequences identify providedantibodies or antigen-binding fragments thereof that bind an epitope inthe extracellular domain of human CD38 (such as aCD38-b-ep), andoptionally also a corresponding epitope of Cynomolgus monkey and/ormurine CD38, either as isolated proteins or on the surface of cellsexpressing CD38 (such as immune cells or cell lines, e.g. Raji cells).

The invention also provides CD38 Modulating Antibody Agents binding thesame (or similar) epitope as bound by the CD38 Modulating AntibodyAgents of the invention. For example, in one embodiment there isprovided an antibody that binds the same (or similar) epitope as(aCD38-b-329 (or variants thereof)).

In some embodiments, the present invention provides anti-CD38 antibodiesor antigen-binding fragments that specifically bind to an epitope ofhuman CD38, wherein the epitope comprises one or more amino acidresidues comprised in the amino acids 65-79 of SEQ ID NO: 9 (i.e.aCD38-b-ep). Preferably the epitope comprises at least 4 amino acidswherein the epitope comprises one or more amino acids comprised in aminoacids 65-79 of SEQ ID NO: 9. Preferably the epitope comprises at least 5amino acids, at least 6 amino acids, at least seven amino acids, atleast eight amino acids, at least nine amino acids, at least ten aminoacids, at least eleven amino acids, at least twelve amino acids, atleast thirteen amino acids, or at least fourteen or more amino acidswherein the epitope comprises one or more amino acids comprised in aminoacids 65-79 of SEQ ID NO: 9. The epitope may be either linear orconformational, i.e. discontinuous. In some embodiments, the anti-CD38antibodies or antigen-binding fragments specifically bind to an epitopeof human CD38 wherein the epitope comprises at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least ten, at least eleven, at leasttwelve, at least thirteen, or at least fourteen or more amino acidresidues comprised in amino acids 65-79 of SEQ ID NO: 9. In someembodiments, the anti-CD38 antibodies or antigen-binding fragments bindto an epitope comprising amino acids 65-79 of SEQ ID NO: 9.

In some embodiments, the present invention provides procedures forscreening and/or characterizing antibodies or antigen-binding fragmentsthereof that comprise a aCD38-b-329 amino acid sequences and/or thatpresent binding features comparable to antibodies or antigen-bindingfragments thereof comprising one or more aCD38-b-329 amino acid sequenceelements (e.g. including aCD38-b-329-HCDR3 amino acid sequence (SEQ IDNO: 3) and/or competing with aCD38-b-329) that allow binding to humanCD38 extracellular domain as isolated protein and on the surface ofcells expressing human CD38, competing for the same epitope inparticular the one identified in the Examples as aCD38-b-ep (proteinsequence ARCVKYTEIHPEMRH; amino acids 65-79 in Uniprot sequence P28907,SEQ ID NO: 9).

Furthermore, the present invention also provides procedures forscreening antibodies or antigen-binding fragments thereof that presentfunctional features comparable to antibodies or antigen-bindingfragments thereof comprising one or more aCD38-b-329 amino acid sequenceelements, such features being cell activating and cytotoxic activities,and acting as CD38 Modulating Antibody Agents. At these scopes, thecandidate antibodies can be tested in the assays that are described inthe Examples (see FIG. 1) or other assays that are is known in the artfor establishing the presence of any of such features, but possibly allof them when evaluated in in vitro/ex vivo assays, cell-based assays,and/or animal models.

In some embodiments, the present invention provides nucleic acidmolecules encoding an isolated antibody or antigen-binding fragmentthereof that comprises a CD38 Modulating Antibody Agent such as aaCD38-b-329 amino acid sequence. In some embodiments, such providednucleic acid molecules may contain codon-optimized nucleic acidsequences, and/or may be included in expression cassettes withinappropriate nucleic acid vectors for the expression in host cells suchas, for example, bacterial, yeast, insect, piscine, murine, simian, orhuman cells.

In some embodiments, the present invention provides host cellscomprising heterologous nucleic acid molecules (e.g. DNA vectors) thatexpress a provided CD38 Modulating Antibody Agent (e.g., an antibody orantigen-binding fragment thereof) having one or more properties, e.g.,as described herein, of a CD38 Modulating Antibody Agent (e.g.,comprising a aCD38-b-329 amino acid sequence). In some embodiments, thepresent disclosure provides methods of preparing a CD38 Modulating Agent(e.g., an antibody or antigen-binding fragment thereof) having one ormore properties, e.g., as described herein, of a CD38 ModulatingAntibody Agent (e.g. comprising a aCD38-b-329 amino acid sequence). Insome embodiments, such methods may comprise culturing a host cell thatcomprises nucleic acids (e.g., heterologous nucleic acids that maycomprise and/or be delivered to the host cell via vectors). In someembodiments, such a host cell (and/or the heterologous nucleic acidsequences) is/are arranged and constructed so that the CD38 ModulatingAntibody Agent (e.g, the antibody or antigen-binding fragment thereof issecreted from the host cell (e.g., so that it can be isolated from cellculture supernatants), and/or exposed on the cell surface (for instance,if such aCD38-b-329 amino acid sequences and sequence elements areintended to be used in the context of, or together with, such cells, asin artificial T cell receptors grafting the specificity of a monoclonalantibody onto T cells).

In some embodiments the antibody or antigen-binding fragment thereof (orvariants of the same) may be afucosylated. It is well known thatantibody glycosylation may have impact on the activity, pharmacokineticsand pharmacodynamics of antibodies (e.g, monoclonal antibodies,recombinant antibodies, and/or antibodies that are otherwise engineeredor isolated) and Fc-fusion proteins and specific technology may beexploited to obtain an antibody with the desired glycosylation profile(Liu L, 2015). Effector functions supporting the cytotoxicity of anantibody for use in accordance with the present invention (e.g., ananti-CD38 antibody as described herein, including for example anantibody which may be or be described as a CD38 Modulating AntibodyAgent) can be enhanced using methods to decrease antibody fucosylationlevels. Antibodies comprising specific aCD38-b-329 sequence elementspresenting such properties can be generated, for example, by expressinga aCD38-b-329 sequence using technologies for genetically engineeringcell lines which may produce antibodies with absent or reducedfucosylation capacity, some of them commercially available such asPotelligent (Lonza) GlyMAXX (ProBiogen), or by manipulating themanufacturing process, for example by controlling osmolarity and/orusing enzyme inhibitors, see also for example the methods described inEP2480671.

In some embodiments, the present invention provides compositions (e.g.pharmaceutical compositions) comprising a provided antibody or anantigen-binding fragment thereof having desirable properties asdescribed herein (e.g., as described for antibodies that are hereintermed CD38 Modulating Antibody Agents, specifically including, forexample, aCD38-b-329 antibodies or antigen-binding fragments thereof,and variants thereof). In some embodiments, such provided compositionsare intended for and/or are use in a medical use, such as a therapeutic,diagnostic, or prophylactic use. In some embodiments, such a providedsuch composition can further comprise a pharmaceutically acceptablecarrier or excipient and/or may be for use in the treatment of cancer.In some embodiments, a pharmaceutical composition may be formulated withone or more carrier, excipients, salts, buffering agents, etc., as isknown in the art. Those of skill in the art will be aware of and readilyable to utilize a variety of formulation technologies, including as maybe particularly desirable and/or useful for a given method and/or siteof administration, for instance for parenteral (e.g. subcutaneous,intramuscular, or intravenous injection), mucosal, intratumoral,peritumoral, oral, or topical administration. In many embodiments,provided pharmaceutical compositions, comprising a CD38 ModulatingAntibody Agent as described herein (e.g., an anti-CD38 antibody orantigen binding portion thereof), are formulated for parenteral delivery(e.g., by injection and/or infusion). In some embodiments, such aprovided pharmaceutical composition may be provided, for example, in apre-loaded syringe or vial format. In some embodiments, such a providedpharmaceutical composition may be provided and/or utilized, for example,in dry (e.g., lyophilized) form; alternatively, in some embodiments,such a provided pharmaceutical composition may be provided and/orutilized in a liquid form (e.g., as a solution, suspension, dispersion,emulsion, etc), in a gel form, etc.

In some embodiments, the present invention provides uses of CD38Modulating Antibody Agents (e.g., anti-CD38 antibodies orantigen-binding fragments thereof as described herein (e.g. comprising aaCD38-b-329 amino acid sequence element), and/or of a compositioncomprising them, in treatment of and/or in the manufacture of amedicament for treatment of, a cancer, such as a B cell malignancy, alymphoma, (Hodgkins Lymphoma, non-Hodgkins lymphoma, chroniclymphocytic, leukemia, acute lymphoblastic leukemia, myelomas), amyeloproliferative disorders, a solid tumor (such as a breast carcinoma,a squamous cell carcinoma, a colon cancer, a head and neck cancer, alung cancer, a genitourinary cancer, a rectal cancer, a gastric cancer,sarcoma, melanoma, an esophageal cancer, liver cancer, testicularcancer, cervical cancer, mastocytoma, hemangioma, eye cancer, laryngealcancer, mouth cancer, mesothelioma, skin cancer, rectal cancer, throatcancer, bladder cancer, breast cancer, uterine cancer, prostate cancer,lung cancer, pancreatic cancer, renal cancer, gastric cancer, non-smallcell lung cancer, and ovarian cancer). The cancer can be also defined onthe basis of presence of specific tumor-relevant markers and antigenssuch as CD20, HER2, PD-1, PD-L1, SLAM7F, CD47, CD137, CD134, TIM3, CD25,GITR, CD38, EGFR, etc., or a cancer that has been identified as having abiomarker referred to as microsatellite instability-high (MSI-H) ormismatch repair deficient (dMMR). Furthermore, such conditions may alsobe considered when defining pre-cancerous, non-invasive states of theabove cancers, such as cancer in-situ, smouldering myeloma, monoclonalgammopathy of undetermined significance, cervical intra-epithelialneoplasia, MALTomas/GALTomes and various lymphoproliferative disorders.Preferably in some embodiments the subject being treated has a solidtumor. In one embodiment the subject has a heamatological cancer. Insome embodiments the subject has a CD38 positive tumor.

Thus, in some embodiments, the present invention provides methods oftreating cancer in a subject, comprising administering to the subject aneffective amount of a composition comprising a provided CD38 ModulatingAntibody Agent (e.g., anti-CD38 antibodies orantigen-binding fragmentsthereof) as described herein (e.g. comprising aCD38-b-329 amino acidsequences). In some embodiments, provided methods may further compriseadministering, simultaneously or sequentially in any order, at least oneadditional agent or therapy to the subject (i.e., so that the subjectreceives a combination therapy). In some embodiments, such an at leastone additional agent or therapy can be or comprise an anticancer drug(e.g., a chemotherapeutic agent, radiotherapy (by applying irradiationexternally to the body or by administering radio-conjugated compounds),an anti-tumor antigen or marker antibody (the antigen or marker beingfor example CD4, CD25, CA125, PSMA, c-MET, VEGF, CD137, VEGFR2, CD20,HER2, HER3, SLAMF7, CD326, CAIX, CD40, CD47, or EGF receptor), acheckpoint inhibitor or an immunomodulating antibody (for example anantibody targeting PD-1. PD-L1, TIM3, CD25, GITR, CD134, CD134L, CD137L,CD80, CD86, B7-H3, B7-H4, B7RP1, LAG3, ICOS, TIM3, GAL9, CD28, AP2M1,SHP-2, OX-40 etc.), a vaccine, an adjuvant, standard-of-use protocol,one or more other compounds targeting cancer cells or stimulating animmune response against cancer cells, or any combination thereof. Incertain particular embodiments, when such at least one additional agentor therapy is or comprises an antibody, the format of and/or the antigentargeted by such antibody can be chosen among those listed in theliterature and possibly adapted to a given cancer (Sliwkowski M &Mellman I, 2013; Redman J M et al., 2015; Kijanka M et al., 2015).

Still further, the present invention provides a variety of kits orarticles of manufacture containing a provided antibody orantigen-binding fragment thereof as described herein (e.g. comprisingaCD38-b-329 amino acid sequences) or related compositions that allow theadministration, storage, or other use of such an isolated antibody orantigen-binding fragment. Preferably the kit comprises a vessel,syringe, a vial, or other container comprising such compositions,optionally together with articles of manufactures, diluents, reagents,solid phases, and/or instructions for the correct use of the kit.

The validation of aCD38-b-329 for medical uses, in particular fortreating cancer, can be performed by performing a more extensiveanalysis of its activity in cell-based assay using differentexperimental set-ups and/or a panel of cancer-derived cell lines.However, given the immunological mechanism associated to CD38 ModulatingAntibody Agent activities, further relevant data can be generated inanimal models wherein cancers are induced or wherein cancer cells areimplanted as a xenograft or as a syngeneic/allogeneic cancer-derivedcells. In addition, these animal models may require the transfer ofhuman cells such as PBMC (i.e. humanized PBMC mice models) or CD34+hematopoietic stem cells (i.e. CD34+ humanized mice) to allow evaluatingactivity of the CD38 Modulating Antibody Agents on human immune cellswithin a mouse system.

Prior to that, the CD38 Modulating Antibody Agent sequences can becloned and expressed into an antibody frame that is more appropriate forpharmaceutical and/or technical reasons. For example, such sequences(possibly as codon-optimized VH and VL coding sequences) can be clonedtogether with human IgG1 constant regions (hIgG1) and expressed using anappropriate antibody expression vectors and cell line (such as aCHO-derived cell line, e.g. CHO-S). Expression and secretion of CD38Modulating Antibody Agent in human IgG1 format antibodies can beanalyzed after transfection in reduced conditions in cell lysates and innon-reduced conditions in supernatants that will be later used to purifythe antibody (by affinity chromatography, gel filtration, and/or otherappropriate technique). The binding and functional properties of CD38Modulating Antibody Agent in human IgG1 format (CD38 Modulating AntibodyAgent-hIgG1) can be analyzed by using the assays described in Examplesbelow. For instance, CD38 Killer Agonist-hIgG1 can be evaluated forbinding to human and cynomolgus PBMC using flow cytometry. Binding tospecific immune cell populations can be assessed in flow cytometrythrough the use of specific markers for specific immune cellpopulations, like CD3, CD45, CD56 and CD159 (NKG2A) for NK cells, CD14(for monocytes), CD19 (for B cells), and/or CD4/CD8 (for T cells).

Moreover, the effect of CD38 Modulating Antibody Agent-hIgG1 antibodieson human primary tumor cells and immune cells isolated from humanhealthy donors and/or patients can be assessed. In order to investigatepotential effects on individual immune cell populations in more detail,CD38 Modulating Antibody Agent-hIgG1 antibodies can be used to treatPBMC, cells isolated from tumors (and/or other organs such as lymphnodes), and/or purified human CD8 and CD4 T cells, Treg cells, MDSCcells, dendritic cells, macrophages and monocytes, neutrophils, NK cellsand other cell types. Potential read outs comprise cytokine release,cell killing, cell proliferation and/or activation, apoptosis,antigen-specific and/or allogeneic responses, or any combinationthereof. Alternatively, mice or non-human primates can be treated andcellular status can be followed using flow cytometry or after isolationof various organs and/or cells from the animals.

Further properties of the CD38 Modulating Antibody Agent-hIgG1antibodies may be evaluated, alone or in combination, by studying theeffects of CD38 Modulating Antibody Agent-hIgG1 antibodies on CD38expressing cells (such as NK or T cells): CD38 enzymatic activity,CD38-induced Ca²+ levels and protein phosphorylation, CD38 sheddingand/or internalization, CD38-induced activation of intra-cellularpathways (e.g. NFkB pathway), and/or interaction with CD31 and otherreceptor proteins (e. g. CD16, TCR, BCR, etc.). The involvement of thelatter processes in the CD38 downstream activity can also be evaluatedusing specific inhibitors of these processes. These cellular effects canthen be followed in vivo when aCD38 Modulating Antibody Agent-hIgG1antibodies are administered to cynomolgus monkeys.

In some embodiments of the invention, the antibodies (and variantsthereof as described herein, such as variants mutated to remove the DGmotif) may have advantageous activity profiles. For example, in oneembodiment, the antibodies or antigen-binding fragments thereof (andvariants of the same) may:

-   -   exhibit antibody-dependent cell-mediated cytotoxicity (ADCC)        activity against CD38+ target cells;    -   exhibits no complement dependent cytotoxicity (CDC) or reduced        CDC activity against a CD38+ target cell as compared to        daratumumab under the same of substantially the same conditions;    -   exhibit antibody-dependent cellular phagocytosis (ADCP); and/or    -   induce immune effector cell activation

Preferably the aCD38-b-329 or antigen binding fragments thereof (orvariants of the same) exhibits, reduced CDC activity against a CD38+target cell as compared to daratumumab under the same or substantiallythe same conditions.

Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of theanti-CD38 antibodies or antigen-binding fragments thereof may bedetermined in vitro using an assay as described in the Examples, e.g.using CD38+ Daudi cells as the target cell and human PBMC cells aseffector cells, wherein the ratio of target cells to effector cells isfrom about 50 to 1 to about 25 to 1.

Complement dependent cytotoxicity (CDC) activity against a CD38+ targetcell can be determined in vitro using an assay as described in theExamples, e.g. using CD38 + Daudi and/or Raji cells in the presence of10% complement. CDC activity may be determined by treating the targetcells with increasing concentrations up to 10 μg/ml of antibodies in thepresence of human complement. In some embodiments CDC activity may bedetermined by measuring the maximum percentage cell lysis of CD38+cells, i.e. CD38+ Daudi cells in the presence of 10% complement. Themaximum lysis for a given antibody may vary between experiments. It istherefore helpful to consider other metrics for measuring CDC activity,including, for example, EC50 values and/or fold difference in maximum %lysis and/or EC50 as compared with a reference antibody (such asdaratumumab). A determination of a lower CDC activity as compared todaratumumab may therefore be in reference to maximum % lysis, EC50,and/or a fold change compared to daratumumab of either value.

In one preferred embodiment of the invention, the CD38 ModulatingAntibody Agents may exhibit may exhibit CDC:

-   -   a) with an EC50 that is at least 0.5-fold higher (or more        preferably at least 1-fold higher) than daratumumab; or    -   b) with a maximum % lysis as measured in Raji and/or Daudi cells        in the presence of 10% complement that is no more than half that        exhibited by daratumumab.

Of course, the CDC of daratumumab is determined in the same orsubstantially the same conditions for the comparison. CDC activity canbe determined using an antibody concentration of up to about 10 μg/mL.As the skilled person would understand, when determining maximum lysisof cells, a concentration of 10 μg/mL is not always required sincemaximum cell lysis may occur at a lower antibody concentration, although10 μg/mL may be used if necessary.

In some embodiments, the reduction in CDC activity compared todaratumumab is such that the EC₅₀ of the antibody or antibody bindingfragment thereof is at least about 0.5-fold greater (i.e. at least about1.5 times greater), or preferably at least about 1-fold greater (i.e. atleast about 2 times greater) than that of daratumumab under the same orsubstantially the same conditions. For example, the EC₅₀ of the antibodyor antibody binding fragment thereof is at least about 0.5 fold greater,or preferably about 1-fold greater than that of daratumumab againstDaudi cells and/or Raji in the presence of 10% complement.

In some embodiments, the antibody or antigen-binding fragment thereof(or variants of the same) induces CDC with an EC₅₀ of at least about0.05 μg/mL against CD38+ Daudi and/or Raji cells (and optionally causesless than 60% lysis of such CD38+ expressing cells by CDC). In someembodiments, the antibody or fragment thereof induces CDC with an EC50of at least about 0.05 μg/mL, at least about 0.10 μg/mL, or at leastabout 0.15 μg/mL against CD38+ Daudi and/or Raji cells (and optionallycauses less than 60% lysis of such CD38+ expressing cells by CDC at anantibody concentration of up to about 10 μg/ml).

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof (or variants of the same) may exhibit antibody-dependentcellular phagocytosis (ADCP) against CD38-expressing cells. ADCPactivity may be determined by a reporter cell assay measuring FcgRllaengagement in Jurkat cells as the effector cells expressing FcgRlla. Theeffector cells also express NFAT-induced luciferase. The target cell inthe assay may be a CD38 expressing Raji cell. NFAT signalling can bemeasured to determine the activity.

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof (or variants of the same) may induce ADCP against in vitrogenerated T reg cells. This may be measured as discussed in theExamples.

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof (or variants of the same) may induce T cell activation at agreater amount as compared to daratumumab under the same orsubstantially the same conditions. In some embodiments T cell activationcan be determined by measuring NFAT signalling in luc_reporter Jurkatcells. In some embodiments, the NFAT signalling induced by the anti-CD38antibody or antigen-binding fragment thereof, as measured inluc_reporter Jurkat cells, is at least about 10% higher than that ofdaratumumab measured under the same or substantially the sameconditions. In some embodiments, the NFAT signalling is at least about15%, at least about 20%, or at least about 30% higher than NFATsignalling of daratumumab measured under the same or substantially thesame conditions.

In a NFAT luc_reporter assay in Jurkat cells, NFAT signalling can bemeasured in the presence of soluble CD3 monoclonal antibody in relativeluminescence units (RLU). The CD3 monoclonal antibody may be at aconcentration of 1 μg/ml and the Jurkat cells may be stimulated with theanti-CD38 antibody at a concentration of from about 5 μg/ml to about 40μg/ml (for example 10 μg/ml). Using such an assay, NFAT signalling maybe at least about 30% higher than NFAT signalling of daratumumabmeasured under the same or substantially the same conditions, when theRLU of CD3 only stimulation is used as a baseline.

T cell activation can be further characterised by an increase in T cellproliferation, and/or an increase in cytokine secretion, wherein thecytokines may be selected from the group consisting of IL-2, TN F-α,IFN-γ, IL-10 and GM-CSF.

T cell proliferation can be measured as in the Examples, for example asdetermined at an antibody concentration of 10 μg/ml after 72 hoursincubation and in the presence of 0.1 μg/ml or 0.5 μg/ml anti-CD3antibody. In some embodiments, the anti-CD38 antibody or antigen-bindingfragment thereof increases T cell proliferation of CD4+ and/or CD8+cells by at least about 20% as compared to untreated cells. In someembodiments, T cell proliferation increases by at least about 25%, by atleast about 30%, by at least about 35%, or by at least about 40% ascompared to untreated cells.

Preferably the anti-CD38 antibody or antigen-binding fragment thereof(or variants of the same) increases T cell proliferation in CD4+ and/orCD8+ cells by at least about 0.5-fold (i.e. at least 1.5 times as much)or at least 1-fold (i.e. at least 2 times as much) or at least 2-fold(i.e. at least 3 times as much) or at least 3-fold (i.e. at least 4times as much) as compared to cells treated with human IgG1 in the sameor substantially the same conditions (for example incubation at the sameantibody concentration for 72 hours).

In some embodiments, the anti-CD38 antibody or antigen-binding fragmentthereof (or variants of the same) induces the secretion of a cytokineselected from the group consisting of IL-2, TNF-α, IFN-γ, IL-10 and/orGM-CSF in CD4+ and/or CD8+ cells in an amount greater than is induced bydaratumumab under the same or substantially the same conditions. In someembodiments, the anti-CD38 antibody or antigen-binding fragment thereofincreases the secretion of GM-CSF as compared to an daratumumab. In someembodiments, the anti-CD38 antibody or antigen-binding fragment thereofincreases the secretion of IL-2 as compared to an daratumumab. In someembodiments, the anti-CD38 antibody or antigen-binding fragment thereofincreases the secretion of IL-2, TNF-α, IFN-γ, IL-10 and GM-CSF ascompared to daratumumab. Cytokine secretion may be measured as providedin the Examples, for example as determined at an antibody concentrationof 10 μg/ml after 72 hours incubation.

As such the anti-CD38 antibodies or antigen-binding fragments thereof(or variants of the same) exhibit antibody-dependent cell-mediatedcytotoxicity (ADCC) activity against CD38+ target cells; exhibit reducedCDC activity against a CD38+ target cell as compared to daratumumabunder the same or substantially the same conditions; induce immuneeffector cell activation; induce T cell proliferation; and induce anincrease in cytokine secretion, including IL-2, IFNγ, TNFα, GM-CSF andIL-10.

The present invention also includes variants or derivates of theantibody aCD38-b-329. Variant or derivative antibodies or antigenbinding fragments thereof (for example, aCD38-b-329-m6 andaCD38-b-329-m7) may share the same functional profile (i.e.pharmacological properties) as for the antibody from which they arederived. Similarly, the present invention includes antibodies or antigenbinding fragments that compete for binding to CD38 with aCD38-b-329 (orvariants thereof). Such competing antibodies may have the samefunctional profile (i.e. pharmacological properties) as aCD38-b-329.

In order to gain further insights into the molecular interactionsbetween the CD38 Modulating Antibody Agent-hIgG1 (e.g. aCD38-b-329-hIgG1antibodies) and human CD38, the crystal structure of theaCD38-b-329-hIgG1 antibody and human CD38 protein can be determined. Thesolubility and stability of the aCD38-b-329-hIgG1 antibodies can beassessed through solubility studies, accelerated stress studies, freezethaw studies and formal stability studies. Aggregation of the antibodiescan be followed by visual inspection, size exclusion chromatography anddynamic light scattering and OD_(280/320) absorbance.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: flowchart summarizing the screening procedure for identifyingaCD38-b-329 as an agonistic anti-CD38 antibody having the one or moreproperties according to the present invention, in particular thosedefining CD38 Modulating Antibody Agents: pharmaceutically relevanttargeted cell killing (e.g. as measured in ADCC, ADCP, and CDC assays),effects on immune cells (such as Treg, CD8 and CD4 T cells, NK cells,dendritic cells, MDSC, macrophages, and/or monocytes, for measuringproperties such as cell viability and/or proliferation, cytokinesecretion, and/or activation markers), effects on CD38 enzymaticactivities or CD38-mediated signaling, effects on cancer cellsexpressing (or not) CD38, combinations with other drugs (e.g. antibodiestargeting a tumor antigen or other anticancer drugs) and/or antibodysequence and format, for identifying stability issues related toaggregation-prone sequences, presence of glycosylation sites or freeCysteines in variable domain and/or effects (e.g. within a human IgG1frame, as Fabs, nanobodies, bi/multispecific antibodies, or withinnon-antibody scaffolds).

FIG. 2: relevant protein sequences (A) aCD38-b-329 protein sequences.Each CDR for the heavy (aCD38-b-329-HCDR1 (SEQ ID NO: 1),aCD38-b-329-HCDR2 (SEQ ID NO: 2), and aCD38-b-329-HCDR3 (SEQ ID NO: 3))and the light (aCD38-b-329-LCDR1 (SEQ ID NO: 5), aCD38-b-329-LCDR2 (SEQID NO: 6), and aCD38-b-329-LCDR3 (SEQ ID NO: 7)) chain is indicatedseparately and, underlined, within the frame sequence of the heavy andlight chain antibody as initially identified by the screening procedure(aCD38-b-329-HCDR123 (SEQ ID NO: 4) and aCD38-b-329-LCDR123(SEQ ID NO:8), respectively). The DG motif (double underlined) is indicate ashotspot for isomerization and degradation of antibodies (Sydow J et al.2014) and may be mutated for providing alternative anti-CD38 antibodieshaving any, and possibly all, binding and functional properties ofaCD38-b-329. (B) sequence of human CD38 (Uniprot code P28907 (SEQ IDNO:9)) in which different boxes identify cytoplasmic domain,transmembrane domain, and, within the extracellular domain, the positionof aCD38-b-329 major epitope as preliminarily identified (aCD38-b-ep)compared to the one of Daratumumab epitope (DARA, as identified anddisclosed in WO2006099875 being formed by two human CD38 regions, hereindicated as DARAep-a and DARAep-b).

FIG. 3: characterization of aCD38-b-329 binding to CD38 expressed inPBMCs, using cells of human origin at increasing antibody concentrationand by restricting the analysis to CD8-positive or CD4-positive cellsand comparing with either human IgG1 isotype control, Daratumumab(DARA), or in absence of a primary antibody.

FIG. 4: functional characterization of aCD38-b-329 compared toDaratumumab (DARA) or negative control antibodies (anti-human CD3 orhuman IgG1 isotype), in cell-based models independently from theadministration of any further tumor targeting antibody. (A) aCD38-b-329increases the percentage of TCR-mediated CD4 and CD8 T cellproliferation, as indicated in each graph. (IgG1 and each of anti-CD38antibodies were tested at 10-5-2.5 μg/ml; anti-CD3 is tested at 0.1μg/ml). (B) aCD38-b-329 increases the secretion of selected cytokines byTCR-activated CD4/CD8 T cells (similar pattern in 5 out of 5 donorstested).

FIG. 5: functional characterization of aCD38-b-329 compared to DARA withrespect to cytotoxicity. (A) This direct antibody-mediated killingeffect is accompanied by antibody-dependent cell-mediated cytotoxicity(ADCC) as in Daratumumab (DARA) but, differently from DARA, not bycomplement-dependent cytotoxicity (CDC, particularly significant forDARA, as described in the literature). (B) Moreover, targets cellkilling, as determined in Daudi cells, is strongly induced by antibodycross-linking when aCD38-b-329 is compared to DARA, whose activity ispoorly increased by antibody cross-linking.

FIG. 6: functional characterization of aCD38-b-329 (administered at 10mg/kg) with respect to animal survival in two cancer models based on theintra-venous administration of Daudi Cells (A) and Ramos cells (B) overthe indicated number of days. The treatment with aCD38-b-329 increasesanimal survival when compared to negative control. In the Daudi modelaCD38-b-329 also increases survival when compared to Daratumumab (DARA).

FIG. 7: Relevant heavy and light chain protein sequences of variantaCD38-b-329 antibodies A) aCD38-b-329-m6 and B) aCD38-b-329-m7. Each CDRfor the heavy and the light chain is underlined, within the framesequence, aCD38-b-329-m6-HCDR123 (SEQ ID NO: 4) andaCD38-b-329-m6-LCDR123 (SEQ ID NO: 16), respectively for aCD38-b-329-m6,and aCD38-b-329-m7-HCDR123 (SEQ ID NO: 4) and aCD38-b-329-m7-LCDR123(SEQ ID NO: 17), respectively for aCD38-b-329-m7.

FIG. 8: Binding of variant sequences to Daudi cells, in comparison withDara and IgG1 control. Binding to CD38 expression human cell line(Daudi) was examined by adding anti-CD38 primary antibodies at 20 μg/mLfollowed by a semi-log dilution series (7 points) followed by stainingwith a secondary antibody. aCD38-b-329 variants show similar binding toDaudi cells as the parental aCD38-b-329 and as DARA.

FIG. 9: Functional characterization of aCD38-b-329 and its variantsequences compared to

DARA with respect to cytotoxicity. aCD38-b-329 and its variant sequencesas well as DARA induce killing of CD38+ cell line (Daudi) byantibody-dependent cell-mediated cytotoxicity (ADCC) with no detectabledifferences in the activity (EC50 or maximum lysis).

FIG. 10: Functional characterization of aCD38-b-329 and variantsaCD38-b-329-m6 and aCD38-b-329-m7 (all administered at 10 mg/kg) withrespect to animal survival in two cancer models based on theintra-venous administration of Raji Cells (A) and Ramos cells (B) overthe indicated number of days. The treatment with aCD38-b-329 increasesanimal survival when compared to negative control in both models. Thetreatment with the variants aCD38-b-329-m6 and a-CD38-b-329-m7 increasesanimal survival when compared to negative control as well as whencompared to Daratumumab (DARA) in both models.

FIG. 11: SPR based analysis of purified antibodies (IgG1) to rhCD38 histagged on the Biacore 2000. A) aCD38-b-329, B) aCD38-b-329-m6 and C)aCD38-b-329-m7,

FIG. 12: Shows the binding of aCD38-b-329 (FIG. 11A) as compared todaratumumab (FIG. 11B) to recombinant human CD38 his tagged measured bybiolayer interferometry on the Octet Red 96 instrument. 4.2 nM ofrhCD38-his was loaded to the Ni NTA biosensor followed by varyingconcentrations of antibody (as shown in the Figures) and then let themto dissociate in Kinetics Buffer.

FIG. 13: Competition Assays in the Octet. Binding of the first antibodyto the immobilized rhCD38 is followed by either the first antibody again(Daratumumab—as control) or a second Ab (aCD38-b-329). Non-competitorantibodies will bind to CD38 in the presence of the other antibody (asshown) while antibodies binding the same epitope will compete, and noadditional binding will be observed.

FIG. 14: shows the binding of anti-CD38 antibody aCD38-b-329 torecombinant human CD38 his tagged measured by biolayer interferometry onthe Octet Red 96 instrument. 4.2 nM of rhCD38-his was loaded to the NiNTA biosensor followed by 8 nM of antibody and then let them todissociate in Kinetics Buffer

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Below are provided certain definitions of terms, technical means, andembodiments used herein, many or most of which confirm commonunderstanding of those skilled in the art.

Administration: As used herein, the term “administration” refers to theadministration of a composition to a subject. Administration to ananimal subject (e.g., to a human) may be by any appropriate route. Forexample, in some embodiments, administration may be bronchial (includingby bronchial instillation), buccal, enteral, intra-arterial,intra-dermal, intra-gastric, intra-medullary, intra-muscular,intra-nasal, intra-peritoneal, intra-thecal, intra-venous,intra-ventricular, within a specific organ or tissue (e. g.intra-hepatic, intra-tumoral, peri-tumoral etc.), mucosal, nasal, oral,rectal, subcutaneous, sublingual, topical, tracheal (including byintra-tracheal instillation), transdermal, vaginal and vitreal. Theadministration may involve intermittent dosing. Alternatively,administration may involve continuous dosing (e.g., perfusion) for atleast a selected period of time. As is known in the art, antibodytherapy is commonly administered parenterally, e.g. by intravenous,subcutaneous, or intratumoral injection (e.g., particularly when highdoses within a tumor are desired).

Agent: The term “agent” as used herein may refer to a compound or entityof any chemical class including, for example, polypeptides, nucleicacids, saccharides, small molecules, metals, or combinations thereof.Specific embodiments of agents that may be utilized in accordance withthe present invention include small molecules, drugs, hormones,antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNAs,shRNAs, antisense oligonucleotides, ribozymes), peptides, peptidemimetics, etc. An agent may be or comprise a polymer.

Antibody: As used herein, the term “antibody” refers to a polypeptidethat includes canonical immunoglobulin sequence elements sufficient toconfer specific binding to a particular target antigen, such as CD38,human CD38 in particular, and human CD38 extracellular domain moreparticularly. As is known in the art, intact antibodies as produced innature are approximately 150 kD tetrameric agents comprised of twoidentical heavy chain polypeptides (about 50 kD each) and two identicallight chain polypeptides (about 25 kD each) that associate with eachother into what is commonly referred to as a “Y-shaped” structure. Eachheavy chain is comprised of at least four domains (each about 110 aminoacids long), an amino-terminal variable (VH) domain (located at the tipsof the Y structure), followed by three constant domains: CH1, CH2, andthe carboxy-terminal CH3 (located at the base of the Y's stem). A shortregion, known as the “switch”, connects the heavy chain variable andconstant regions. The “hinge” connects CH2 and CH3 domains to the restof the antibody. Two disulfide bonds in this hinge region connect thetwo heavy chain polypeptides to one another in an intact antibody. Eachlight chain is comprised of two domains—an amino-terminal variable (VL)domain, followed by a carboxy-terminal constant (CL) domain, separatedfrom one another by another “switch”. Intact antibody tetramers arecomprised of two heavy chain-light chain dimers in which the heavy andlight chains are linked to one another by a single disulfide bond; twoother disulfide bonds connect the heavy chain hinge regions to oneanother, so that the dimers are connected to one another and thetetramer is formed. Naturally produced antibodies are also glycosylated,typically on the CH2 domain, and each domain has a structurecharacterized by an “immunoglobulin fold” formed from two beta sheets(e.g., 3-, 4-, or 5-stranded sheets) packed against each other in acompressed antiparallel beta barrel. Each variable domain contains threehypervariable loops known as “complement determining regions” (CDR1,CDR2, and CDR3; as understood in the art, for example determinedaccording to Kabat numbering scheme) and four somewhat invariant“framework” regions (FR1, FR2, FR3, and FR4). When natural antibodiesfold, the FR regions form the beta sheets that provide the structuralframework for the domains, and the CDR loop regions from both the heavyand light chains are brought together in three-dimensional space so thatthey create a single hypervariable antigen-binding site located at thetip of the Y structure. The Fc region of naturally-occurring antibodiesbinds to elements of the complement system, and also to receptors oneffector cells, including for example effector cells that mediatecytotoxicity. As is known in the art, affinity and/or other bindingattributes of Fc regions for Fc receptors can be modulated throughglycosylation or other modification that can improve the developabilityof the antibody (Jarasch A et al., 2015).

In some embodiments, antibodies produced and/or utilized in accordancewith the present invention include glycosylated Fc domains, including Fcdomains with modified or engineered such glycosylation. For purposes ofthe present invention, in certain embodiments, any polypeptide orcomplex of polypeptides that includes sufficient immunoglobulin domainsequences as found in natural antibodies can be referred to and/or usedas an “antibody”, whether such polypeptide is naturally produced (e.g.,generated by an organism reacting to an antigen), or produced byrecombinant engineering, chemical synthesis, or other artificial systemor methodology. In some embodiments, an antibody is polyclonal oroligoclonal, that is generated as a panel of antibodies, each associatedto a single antibody sequence and binding a more or less distinctepitopes within an antigen (such as different epitopes within human CD38extracellular domain that are associated to different referenceanti-CD38 antibodies).

Polyclonal or oligoclonal antibodies can be provided in a singlepreparation for medical uses as described in the literature (Kearns J Det al., 2015). In some embodiments, an antibody is monoclonal. In someembodiments, an antibody has constant region sequences that arecharacteristic of mouse, rabbit, primate, or human antibodies. In someembodiments, antibody sequence elements are humanized, primatized,chimeric, etc, as is known in the art. Moreover, the term “antibody” asused herein, can refer in appropriate embodiments (unless otherwisestated or clear from context) to any of the art-known or developedconstructs or formats for utilizing antibody structural and functionalfeatures in alternative presentation, for instance as antigen-bindingfragments as defined below. For example, an antibody utilized inaccordance with the present invention is in a format selected from, butnot limited to, intact IgG, IgE and IgM, bi- or multi-specificantibodies (e.g., Zybodies®, etc), single chain variable domains (scFv),polypeptide-Fc fusions, Fabs, cameloid antibodies, heavy-chain sharkantibody (IgNAR), masked antibodies (e.g., Probodies®), or fusionproteins with polypeptides that allow expression and exposure on thecell surface (as scFv within constructs for obtaining artificial T cellreceptors that are used to graft the specificity of a monoclonalantibody onto a T cell). In some embodiments, an antibody may lack acovalent modification (e.g., attachment of a glycan) that it would haveif produced naturally. Alternatively, an antibody may contain a covalentmodification (e.g., attachment of a glycan, a payload [e.g., adetectable moiety, a therapeutic moiety, a catalytic moiety, etc.], orother pendant group [e.g., poly-ethylene glycol, etc.]).

Antigen: The term “antigen”, as used herein, refers to an agent thatelicits an immune response and/or that binds to a T cell receptor (e.g.,when presented by an MHC molecule) and/or B cell receptor. An antigenthat elicits a humoral response involve the production ofantigen-specific antibodies or, as shown in the Examples for CD38extracellular domain, can be used for screening antibody libraries andidentifying candidate antibody sequences to be further characterized.

Antigen-binding Fragment: As used herein, the term “Antigen-bindingFragment” encompasses agents that include or comprise one or moreportions of an antibody as described herein sufficient to confer on theantigen-binding fragment and ability to specifically bind to the Antigentargeted by the antibody. For example, in some embodiments, the termencompasses any polypeptide or polypeptide complex that includesimmunoglobulin structural elements sufficient to confer specificbinding. Exemplary antigen-binding fragments include, but are notlimited to Small Modular ImmunoPharmaceuticals (“SMIPs™”), single chainantibodies, cameloid antibodies, single domain antibodies (e.g., sharksingle domain antibodies), single chain or Tandem diabodies (TandAb®),VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeatproteins or DARPINs®, Avimers®, a DART, a TCR-like antibody, Adnectins®,Affilins®, Trans-bodies®, Affibodies®, a TrimerX®, MicroProteins,Centyrins®, CoVX bodies, BiCyclic peptides, Kunitz domain derivedantibody constructs, or any other antibody fragments so long as theyexhibit the desired biological activity. In some embodiments, the termencompasses other protein structures such as stapled peptides,antibody-like binding peptidomimetics, antibody-like binding scaffoldproteins, monobodies, and/or other non-antibody proteins scaffold, forexample as reviewed in the literature (Vazquez-Lombardi R et al., 2015).In some embodiments, an antigen-binding fragment is or comprises apolypeptide whose amino acid sequence includes one or more structuralelements recognized by those skilled in the art as a complementaritydetermining region (CDR). In some embodiments an antigen-bindingfragment is or comprises a polypeptide whose amino acid sequenceincludes at least one reference CDR (e.g., at least one heavy chain CDRand/or at least one light chain CDR) that is substantially identical toone found in an anti-CD38 antibody as described herein (e.g., in anaCD38-b-329 amino acid sequence element), and in particular at least oneheavy chain CDR, such as an HCDR3 (e.g., an aCD38-b-329-HCDR3 sequence).In some embodiments, an antigen-binding fragment is or comprises apolypeptide whose amino acid sequence includes at least one CDR (e.g.,at least one heavy chain CDR and/or at least one light chain CDR) thatis either identical in sequence or contains a small number (e.g., 1, 2,3, or) or more amino acid alterations (e.g., substitutions, additions,or deletions; in many cases, substitutions) relative to such a referenceCDR, while maintaining binding to the target of the antibody (e.g.,aCD38-b-329) from which the reference CDR was derived. In someembodiments, an antigen-binding fragment is or comprises a polypeptideor complex thereof that includes all three CDRs (or, in someembodiments, sequences substantially identical thereto) from a heavy orlight chain of a reference antibody (e.g., from aCD38-b-329); in someembodiments, an antigen-binding fragment is or comprises a polypeptideor complex thereof that includes all six CDRs (or, in some embodiments,sequences substantially identical thereto) from a reference antibody(e.g., from aCD38-b-329). In some embodiments, an antigen-bindingfragment is or comprises a polypeptide or complex thereof that includesthe heavy and/or light chain variable domains (or, in some embodiments,sequences substantially identical thereto) of a reference antibody(e.g., of aCD38-b-329). In some embodiments, the term “antigen-bindingfragment” encompasses non-peptide and non-protein structures, such asnucleic acid aptamers, for example, RNA aptamers and DNA aptamers. Anaptamer is an oligonucleotide (e.g., DNA, RNA, or an analog orderivative thereof) that binds to a particular target, such as apolypeptide. Aptamers are short synthetic single-strandedoligonucleotides that specifically bind to various molecular targetssuch as small molecules, proteins, nucleic acids, and even cells andtissues. These small nucleic acid molecules can form secondary andtertiary structures capable of specifically binding proteins or othercellular targets, and are essentially a chemical equivalent ofantibodies. Aptamers are highly specific, relatively small in size, andnon-immunogenic. Aptamers are generally selected from a biopanningmethod known as SELEX (Systematic Evolution of Ligands by Exponentialenrichment) (See for example Ellington et al. Nature. 1990; 346(6287):818-822; Tuerk et al., Science. 1990; 249(4968):505-510; Ni et al., CurrMed Che 2011; 18(27):4206-14). Methods of generating an apatmer for anygiven target are well known in the art. Peptide aptamers includingaffimers are also encompassed. An affimer is a small, highly stableprotein engineered to display peptide loops which provide a highaffinity binding surface for a specific target protein. It is a proteinof low molecular weight, 12-14 kDa, derived from the cysteine proteaseinhibitor family of cystatins. Affimer proteins are composed of ascaffold, which is a stable protein based on the cystatin protein fold.They display two peptide loops and an N-terminal sequence that can berandomized to bind different target proteins with high affinity andspecificity similar to antibodies. Stabilization of the peptide upon theprotein scaffold constrains the possible conformations which the peptidemay take, thus increasing the binding affinity and specificity comparedto libraries of free peptides.

Percent (%) sequence identity: Percent (%) “sequence identity” betweentwo sequences can be determined using those methods known in the art.Sequence identity with respect to a peptide, polypeptide or antibodysequence can be defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, includinggapped BLAST, and BLASTp (for proteins), (Altschul S F et al (1997)), orFASTA., using the default parameters.

Biological Sample. As used herein, the terms “biological sample” or”“sample” typically refers to a sample obtained or derived from abiological source (e.g., a tissue or organism or cell culture) ofinterest, as described herein. A source of interest may be an organism,such as an animal or human. The biological sample may comprisebiological tissue or fluid.

Cancer. The terms “cancer”, “malignancy”, “neoplasm”, “tumor”, “tumour”,and “carcinoma”, are used interchangeably herein to refer to cells thatexhibit relatively abnormal, uncontrolled, and/or autonomous growth, sothat they exhibit an aberrant growth phenotype characterized by asignificant loss of control of cell proliferation. In general, cells ofinterest for detection or treatment in the present application includeprecancerous (e.g., benign), malignant, pre-metastatic, metastatic, andnon-metastatic cells. The teachings of the present disclosure may berelevant to any and all cancers. To give but a few, non-limitingexamples, in some embodiments, teachings of the present disclosure areapplied to one or more cancers such as, for example, hematopoieticcancers including leukemias, lymphomas (Hodgkins and non-Hodgkins),myelomas and myeloproliferative disorders; sarcomas, melanomas,adenomas, carcinomas of solid tissue, squamous cell carcinomas of themouth, throat, larynx, and lung, liver cancer, genitourinary cancerssuch as prostate, cervical, bladder, uterine, and endometrial cancer andrenal cell carcinomas, bone cancer, pancreatic cancer, skin cancer,cutaneous or intraocular melanoma, cancer of the endocrine system,cancer of the thyroid gland, cancer of the parathyroid gland, head andneck cancers, breast cancer, gastro-intestinal cancers and nervoussystem cancers, benign lesions such as papillomas, and the like. Theantibodies of the invention can be used in the treatment of CD38+expressing tumors.

CD38 Modulating Antibody Agent The term “CD38 Modulating Antibody Agent”is used herein to refer to those CD38 Modulating Antibody Agents (e.g.,anti-CD38 antibodies) that demonstrate particular properties asdescribed herein. In many embodiments, desirable CD38 ModulatingAntibody Agents as described herein are characterized in that theystimulate immune effector cells and/or modify immune cells function andare cytotoxic towards or induce phagocytosis of CD38 expressing cells(e.g. expressing high levels of CD38) such as immune suppressive cellsor tumour cells (e.g., in each case, that express CD38 on theirsurfaces),In some embodiments a CD38 Modulating Antibody Agent ischaracterized by an activity (e.g., level and/or type) reasonablycomparable to that of aCD38-b-329 with respect to immune cells (e.g.,when contacted with immune cells, and particularly with immune cellsthat express CD38) and tumour cells. In some embodiments, a relevantactivity is or comprises ADCP, ADCC in absence of CDC, direct killing,depletion of certain CD38-expressing cells (e.g., high-expressingcells), effector immune cell activation, promotion of T cell, B cell orNK cell expansion, modulation of immune cells activity (e.g.repolarization of suppressive macrophages into inflammatorymacrophages), skewing of T cell repertoire, etc., and combinationsthereof. In some embodiments, CD38 Modulating Antibody Agents areentities or moieties whose presence or level correlates with leveland/or activity of CD38, and/or with one or more features or resultscharacteristic of CD38 activity. In some embodiments, an increased leveland/or activity is assessed or determined relative to that observedunder otherwise comparable conditions in absence of the entity(ies) ormoiety(ies). Alternatively or additionally, in some embodiments, anincreased level and/or activity is comparable to or greater than thatobserved under comparable conditions when a reference CD38 ModulatingAntibody Agent (e.g., an appropriate reference anti-CD38 antibody, whichin many embodiments is a CD38 agonist antibody, such as IB4) is present.In many embodiments, a CD38 Modulating Antibody Agent for use inaccordance with the present disclosure is or comprises an entity ormoiety that binds, directly or indirectly, to CD38, typically to itsextracellular domain. In some embodiments, a CD38 Modulating AntibodyAgent is, comprises, or competes for binding to CD38 with an anti-CD38antibody as exemplified herein, an antigen-binding fragment (e.g.,comprising one or more CDRs, all heavy chain CDRs, all light chain CDRs,all CDRs, a heavy chain variable region, a light chain variable region,or both heavy and light chain variable regions) thereof, an affinitymatured variant thereof (or an antigen-binding fragment thereof), or anyalternative format (e.g., chimeric, humanized, multispecific, alternateisotype, etc) of any of the foregoing. Alternatively or additionally, insome embodiments, a CD38 Modulating Antibody Agent as described hereinmay be characterized by one or more features that may be features thatare advantageous for screening, manufacturing, (pre-)clinical testing,and/or identifying relevant epitope within human CD38, (such as thesequence identified as aCD38-b-ep), and/or for formulation,administration, and/or efficacy in particular contexts (e.g., for cancertherapy), as disclosed herein.

Combination Therapy: As used herein, the term “combination therapy”refers to those situations in which a subject is simultaneously exposedto two or more therapeutic regimens (e.g., two or more therapeuticagents). In some embodiments, two or more agents may be administeredsimultaneously. Alternatively, such agents may be administeredsequentially; otherwise, such agents are administered in overlappingdosing regimens.

Comparable: As used herein, the term “comparable” refers to two or moreagents, entities, situations, effects, sets of conditions, etc., thatmay not be identical to one another but that are sufficiently similar topermit comparison (e.g., by level and/or type of activity) there betweenso that conclusions may reasonably be drawn based on differences orsimilarities observed. Such comparable sets of conditions, effects,circumstances, individuals, or populations are characterized by aplurality of substantially identical features and one or a small numberof varied features. Those of ordinary skill in the art will understand,in context, what degree of identity is required in any givencircumstance for two or more such agents, entities, situations, sets ofconditions, effects, or populations, etc. to be considered comparable.

Comprising: A composition or method described herein as “comprising” oneor more named elements or steps is open-ended, meaning that the namedelements or steps are essential, but other elements or steps may beadded within the scope of the composition or method. It is alsounderstood that any composition or method described as “comprising” (orwhich “comprises”) one or more named elements or steps also describesthe corresponding, more limited composition or method “consistingessentially of” (or which “consists essentially of”) the same namedelements or steps, meaning that the composition or method includes thenamed essential elements or steps and may also include additionalelements or steps that do not materially affect the basic and novelcharacteristic(s) of the composition or method.

Daratumumab: As used herein, the term “daratumumab” includes an antibodyhaving, VH and VL sequences as published in WO2006/099875 and being ahuman IgG1 monoclonal antibody. For example having variable heavy andlight chain sequences comprising the sequences as provided below:

Heavy Chain: (SEQ ID NO: 18)EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K Light Chain(SEQ ID NO: 19) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Dosage Form: As used herein, the term “dosage form” refers to aphysically discrete unit of an active agent (e.g., a therapeutic ordiagnostic agent) for administration to a subject. Each unit contains apredetermined quantity of active agent. In some embodiments, suchquantity is a unit dosage amount (or a whole fraction thereof)appropriate for administration in accordance with a dosing regimen thathas been determined to correlate with a desired or beneficial outcomewhen administered to a relevant population (i.e., with a therapeuticdosing regimen). Those of ordinary skill in the art appreciate that thetotal amount of a therapeutic composition or agent administered to aparticular subject is determined by one or more attending physicians andmay involve administration of multiple dosage forms.

Dosing Regimen: As used herein, the term “dosing regimen” refers to aset of unit doses (typically more than one) that are administeredindividually to a subject, typically separated by periods of time. Insome embodiments, a given therapeutic agent has a recommended dosingregimen, which may involve one or more doses. In some embodiments, adosing regimen comprises a plurality of doses each of which areseparated from one another by a time period of the same length.Alternatively, a dosing regimen comprises a plurality of doses and atleast two different time periods separating individual doses. In someembodiments, all doses within a dosing regimen are of the same unit doseamount. Alternatively, different doses within a dosing regimen are ofdifferent amounts. In some embodiments, a dosing regimen comprises afirst dose in a first dose amount, followed by one or more additionaldoses in a second dose amount different from the first dose amount. Adosing regimen may comprise a first dose in a first dose amount,followed by one or more additional doses in a second dose amount same asthe first dose amount. In some embodiments, a dosing regimen iscorrelated with a desired or beneficial outcome when administered acrossa relevant population (i.e., is a therapeutic dosing regimen).

Epitope: As used herein, the term “epitope” refers to a portion of anantigen that is bound by an antibody or antigen-binding fragment. Insome embodiments, where the antigen is a polypeptide, an epitope isconformational in that it is comprised of portions of an antigen thatare not covalently contiguous in the antigen but that are near to oneanother in three-dimensional space when the antigen is in a relevantconformation. For example, for CD38, conformational epitopes are thosecomprised of amino acid residues that are not contiguous in CD38extracellular domain; linear epitopes are those comprised of amino acidresidues that are contiguous in CD38 extracellular domain. In someembodiments, epitopes utilized in accordance with the present inventionare provided by means of reference to those bound by CD38 ModulatingAntibody Agents provided herein (e.g., by aCD38-b-329 and defined asaCD38-b-ep). Means for determining the exact sequence and/orparticularly amino acid residues of the epitope for aCD38-b-329 areknown in the literature and in the Examples, including competition withpeptides, from antigen sequences, binding to CD38 sequence fromdifferent species, truncated, and/or mutagenized (e.g. by alaninescanning or other site-directed mutagenesis), phage display-basedscreening, or (co-) crystallography techniques.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition is or may be administered,e.g., for experimental, diagnostic, prophylactic, cosmetic, and/ortherapeutic purposes. Typical patients include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and/or humans). In someembodiments, a patient is a human. In some embodiments, a patient issuffering from or susceptible to one or more disorders or conditions. Apatient may display one or more symptoms of a disorder or condition, ormay have been diagnosed with one or more disorders or conditions (suchas cancer, or presence of one or more tumors). In some embodiments, thepatient is receiving or has received certain therapy to diagnose and/orto treat such disease, disorder, or condition.

Pharmaceutically Acceptable: As used herein, the term “pharmaceuticallyacceptable” applied to the carrier, diluent, or excipient used toformulate a composition as disclosed herein means that the carrier,diluent, or excipient must be compatible with the other ingredients ofthe composition and not deleterious to the recipient thereof.

Pharmaceutical Composition: As used herein, the term “pharmaceuticalcomposition” refers to a composition in which an active agent isformulated together with one or more pharmaceutically acceptablecarriers. In some embodiments, active agent is present in unit doseamount appropriate for administration in a therapeutic regimen thatshows a statistically significant probability of achieving apredetermined therapeutic effect when administered to a relevantpopulation. A pharmaceutical compositions may be formulated foradministration in solid or liquid form, including those adapted for thefollowing: oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; parenteral administration, forexample, by subcutaneous, intramuscular, intravenous, intratumoral, orepidural injection as a sterile solution or suspension, orsustained-release formulation; topical application, for example, as acream, ointment, or a controlled-release patch or spray applied to skin,lungs, or oral cavity; intravaginally, intrarectally, sublingually,ocularly, transdermally, nasally, pulmonary, and to other mucosalsurfaces.

Solid Tumor. As used herein, the term “solid tumor” refers to anabnormal mass of tissue that usually does not contain cysts or liquidareas. Solid tumors may be benign or malignant. Different types of solidtumors are named for the type of cells that form them. Examples of solidtumors are sarcomas (including cancers arising from transformed cells ofmesenchymal origin in tissues such as cancellous bone, cartilage, fat,muscle, vascular, hematopoietic, or fibrous connective tissues),carcinomas (including tumors arising from epithelial cells), melanomas,lymphomas, mesothelioma, neuroblastoma, retinoblastoma, etc. Cancersinvolving solid tumors include, without limitations, brain cancer, lungcancer, stomach cancer, duodenal cancer, esophagus cancer, breastcancer, colon and rectal cancer, renal cancer, bladder cancer, kidneycancer, pancreatic cancer, prostate cancer, ovarian cancer, melanoma,mouth cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer,vaginal cancer, neck cancer, lymphoma, and the like.

Therapeutically Effective Amount: As used herein, the term“therapeutically effective amount” means an amount (e.g., of an agent orof a pharmaceutical composition) that is sufficient, when administeredto a population suffering from or susceptible to a disease and/orcondition in accordance with a therapeutic dosing regimen, to treat suchdisease and/or condition. A therapeutically effective amount is one thatreduces the incidence and/or severity of, stabilizes, and/or delaysonset of, one or more symptoms of the disease, disorder, and/orcondition. Those of ordinary skill in the art will appreciate that a“therapeutically effective amount” does not in fact require successfultreatment be achieved in a particular subject.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a substance (e.g., aprovided CD38 Modulating Antibody Agent, as exemplified by aCD38-b-329,or any other agent) that partially or completely alleviates,ameliorates, relives, inhibits, delays onset of, reduces severity of,and/or reduces incidence of one or more symptoms. In some embodiments,treatment may involve the direct administration of a CD38 ModulatingAntibody Agent such as aCD38-b-329 (for example, as an injectable,aqueous composition, optionally comprising a pharmaceutically acceptablecarrier, excipient and/or adjuvant, for use for intravenous,intratumoral or peritumoral injection) or the administration using aregimen comprising obtaining cells from the subject (e.g. from theblood, a tissue, or a tumor, with or without a selection on the basis ofpresence, or absence, of the expression of a marker), contacting saidcells with a CD38 Modulating Antibody Agent such as aCD38-b-329 ex vivo,and administering such cells to the subject (with or without a selectionon the basis of presence, or absence, of the expression of a marker).

Dosing and Administration. Pharmaceutical compositions comprising a CD38Modulating Agent as described herein (.e.g, an anti-CD38 orantigen-binding fragment thereof, for example, comprising theaCD38-b-329-HCDR3 amino acid sequence for use in accordance with thepresent invention may be prepared for storage and/or delivery using anyof a variety of techniques and/or technologies known and/or available tothose skilled in the art. In some embodiments, a provided CD38Modulating Antibody Agent is administered according to a dosing regimenapproved by a regulatory authority such as the United States Food andDrug Administration (FDA) and/or the European Medicines Agency (EMEA),e.g., for the relevant indication. In some embodiments, a provided CD38Modulating Antibody Agent is administered in combination with one ormore other agents or therapies, which may themselves be administeredaccording to a dosing regimen approved by a regulatory authority such asthe United States Food and Drug Administration (FDA) and/or the EuropeanMedicines Agency (EMEA), e.g., for the relevant indication. In someembodiments however, use of a provided CD38 Modulating Agent may permitreduced dosing (e.g., lower amount of active in one or more doses,smaller number of doses, and/or reduced frequency of doses) of anapproved agent or therapy used in combination with the CD38 ModulatingAntibody Agent therapy. The dosing and administration may be alsoadapted to other drugs that also administered, the patient status,and/or the format of CD38 Modulating Antibody Agent (e.g. modified as animmunoconjugate, a nanobody, or a bispecific antibody).

Moreover, in some embodiments, it may be desirable to tailor dosingregimens, and particularly to design sequential dosing regimens, basedon timing and/or threshold expression levels of CD38, whether forparticular cell types, particular tumors or types thereof, or particularpatient populations (e.g., carrying genetic markers). In some suchembodiments, therapeutic dosing regimens may be combined with oradjusted in light of detection methods that assess expression of one ormore inducible markers or other criteria prior to and/or during therapy.

In some embodiments, dosing and administration according to the presentinvention utilizes active agent having a desired degree of puritycombined with one or more physiologically acceptable carriers,excipients or stabilizers in any or variety of forms. These include, forexample, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Apreferred form may depend on the intended mode of administration and/ortherapeutic application, typically in the form of injectable orinfusible solutions, such as compositions similar to those used fortreating of human subjects with antibodies.

In some embodiments, ingredient(s) can be prepared with carriers thatprotect the agent(s) against rapid release and/or degradation, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as polyanhydrides, polyglycolic acid,polyorthoesters, and polylactic acid. In general, each active agent isformulated, dosed, and administered in therapeutically effective amountusing pharmaceutical compositions and dosing regimens that areconsistently with good medical practice and appropriate for the relevantagent(s) (e.g., for agents such as antibodies). Pharmaceuticalcompositions containing active agents can be administered by anyappropriate method known in the art, including, without limitation,oral, mucosal, by-inhalation, topical, buccal, nasal, rectal, orparenteral (e.g. intravenous, infusion, intratumoral, intranodal,subcutaneous, intraperitoneal, intramuscular, intradermal, transdermal,or other kinds of administration involving physical breaching of atissue of a subject and administration of the pharmaceutical compositionthrough such breach).

In some embodiments, a dosing regimen for a particular active agent mayinvolve intermittent or continuous (e.g., by perfusion or slow releasesystem) administration, for example to achieve a particular desiredpharmacokinetic profile or other pattern of exposure in one or moretissues or fluids of interest in the subject. In some embodiments,different agents administered in combination may be administered viadifferent routes of delivery and/or according to different schedules.Alternatively or additionally, in some embodiments, one or more doses ofa first active agent is administered substantially simultaneously with,and in some embodiments via a common route and/or as part of a singlecomposition with, one or more other active agents.

Factors to be considered when optimizing routes and/or dosing schedulefor a given therapeutic regimen may include, for example, the particularcancer being treated (e.g., type, stage, location, etc.), the clinicalcondition of a subject (e.g., age, overall health, weight, etc.), thesite of delivery of the agent, the nature of the agent (e.g. an antibodyor other protein-based compound), the mode and/or route ofadministration of the agent, the presence or absence of combinationtherapy, and other factors known to medical practitioners.

Those skilled in the art will appreciate, for example, that a specificroute of delivery may impact dose amount and/or required dose amount mayimpact route of delivery. For example, where particularly highconcentrations of an agent within a particular site or location (e.g.,within an tissue or organ) are of interest, focused delivery (e.g.,intratumoral delivery) may be desired and/or useful. In someembodiments, one or more features of a particular pharmaceuticalcomposition and/or of a utilized dosing regimen may be modified overtime (e.g., increasing or decreasing amount of active in any individualdose, increasing or decreasing time intervals between doses, etc.), forexample in order to optimize a desired therapeutic effect or response(e.g., a therapeutic or biological response that is related to thefunctional features of a CD38, Modulating Antibody Agent as describedherein). In general, type, amount, and frequency of dosing of activeagents in accordance with the present invention in governed by safetyand efficacy requirements that apply when relevant agent(s) is/areadministered to a mammal, preferably a human. In general, such featuresof dosing are selected to provide a particular, and typicallydetectable, therapeutic response as compared with what is observedabsent therapy. In context of the present invention, an exemplarydesirable therapeutic response may involve, but is not limited to,inhibition of and/or decreased tumor growth, tumor size, metastasis, oneor more of the symptoms and side effects that are associated with thetumor, as well as increased apoptosis of cancer cells, therapeuticallyrelevant decrease or increase of one or more cell marker or circulatingmarkers and the like. Such criteria can be readily assessed by any of avariety of immunological, cytological, and other methods that aredisclosed in the literature. For example, the therapeutically effectiveamount of CD38 Modulating Antibody Agent, alone or in combination with afurther agent, can be determined as being sufficient to enhance killingof cancer cells as described in the Examples.

A therapeutically effective amount of a CD38 Modulating Antibody Agentas active agent or composition comprising such agent can be readilydetermined using techniques available in the art including, for example,considering one or more factors such as the disease or condition beingtreated, the stage of the disease, the age and health and physicalcondition of the mammal being treated, the severity of the disease, theparticular compound being administered, and the like.

In some embodiments, therapeutically effective amount is an effectivedose (and/or a unit dose) of an active agent that may be at least about0.01 μg/kg body weight, at least about 0.05 μg/kg body weight; at leastabout 0.1 μg/kg body weight, at least about 1 μg/kg body weight, atleast about 5 μg/kg body weight, at least about 10 μg/kg body weight, ormore (e.g. about 100 μg/kg body weight). It will be understood by one ofskill in the art that in some embodiments such guidelines may beadjusted for the molecular weight of the active agent. The dosage mayalso be varied for route of administration, the cycle of treatment, orconsequently to dose escalation protocol that can be used to determinethe maximum tolerated dose and dose limiting toxicity (if any) inconnection to the administration of the isolated antibody orantigen-binding fragment thereof comprising the aCD38-b-329-HCDR3 aminoacid sequence at increasing doses.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high drug concentration. Sterileinjectable solutions can be prepared by incorporating the antibody inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and otherrequired ingredients from those enumerated above. In the case of powdersfor preparing sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze drying that yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile filtered solution. The proper fluidity of a solutioncan be maintained, for example, by using a coating, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. Prolonged absorption of injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, monostearate salts and gelatin.

The formulation of each agent should desirably be sterile, as can beaccomplished by filtration through sterile filtration membranes, andthen packaged, or sold in a form suitable for bolus administration orfor continuous administration. Injectable formulations may be prepared,packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Formulations for parenteraladministration include, but are not limited to, suspensions, solutions,emulsions in oily or aqueous vehicles, pastes, and implantablesustained-release or biodegradable formulations as discussed herein.Sterile injectable formulations may be prepared using a non-toxicparenterally acceptable diluent or solvent, such as water or 1,3butanediol. Other parentally-administrable formulations which are usefulinclude those which comprise the active ingredient in microcrystallineform, in a liposomal preparation, or as a component of biodegradablepolymer systems. Compositions for sustained release or implantation maycomprise pharmaceutically acceptable polymeric or hydrophobic materialssuch as an emulsion, an ion exchange resin, a sparingly soluble polymeror salt.

Each pharmaceutical composition for use in accordance with the presentinvention may include pharmaceutically acceptable dispersing agents,wetting agents, suspending agents, isotonic agents, coatings,antibacterial and antifungal agents, carriers, excipients, salts, orstabilizers are non-toxic to the subjects at the dosages andconcentrations employed. A non-exhaustive list of such additionalpharmaceutically acceptable compounds includes buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; salts containing pharmacologicallyacceptable anions (such as acetate, benzoate, bicarbonate, bisulfate,isothionate, lactate, lactobionate, laurate, malate, maleate,salicylate, stearate, subacetate, succinate, tannate, tartrate,teoclate, tosylate, thiethiodode, and valerate salts); preservatives(such as octadecyidimethylbenzyl ammonium chloride; hexamethoniumchloride; benzalkonium chloride, benzethonium chloride; sodium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

In some embodiments, where two or more active agents are utilized inaccordance with the present invention, such agents can be administeredsimultaneously or sequentially. In some embodiments, administration ofone agent is specifically timed relative to administration of anotheragent. In some embodiments, desired relative dosing regimens for agentsadministered in combination may be assessed or determined empirically,for example using ex vivo, in vivo and/or in vitro models; in someembodiments, such assessment or empirical determination is made in vivo,in a particular patient or patient population (e.g., so that acorrelation is made).

In some embodiments, one or more active agents utilized in practice ofthe present invention is administered according to an intermittentdosing regimen comprising at least two cycles. Where two or more agentsare administered in combination, and each by such an intermittent,cycling, regimen, individual doses of different agents may beinterdigitated with one another. In some embodiments, one or more dosesof the second agent is administered a period of time after a dose of aCD38 Modulating Antibody Agent as described herein. In some embodiments,each dose of the second agent is administered a period of time after adose of CD38 Modulating Antibody Agent as described herein. In someembodiments, a CD38 Modulating Antibody Agent as described herein can bealso administered in regimens that involve not only subsequentadministration by the same route but also by alternating administrationroutes such as by sub-cutaneous (or intramuscular) administration andintra-tumoral administration, within one or more cycles of treatmentsover one, two, four or more weeks, repeating such cycle with the sameregimen (or by extending the interval between administrations),depending of patient responses. Also, in some embodiments, the preciseregimen followed (e.g., number of doses, spacing of doses (e.g.,relative to each other or to another event such as administration ofanother therapy), amount of doses, etc. may be different for one or morecycles as compared with one or more other cycles.

By using any of the routes of administrations, dosages, and/or regimensas described herein, a CD38 Modulating Antibody Agent as describedherein can be identified, characterized and/or validated, for example,taking into account one or more criteria that are measured in thepatients using biopsies, blood samples, and/or other clinical criteria.In some embodiments, as an alternative or in addition direct evaluationof tumor size and/or metastasis, therapeutic efficacy of a CD38Modulating Antibody Agent as described herein can be determined inmethods wherein one or more different general criteria are evaluated:direct cytotoxicity on cancer cells (apoptosis and necrosis of cancercells), increase of tumor infiltrating, immune cells (such asCD4-positive and/or CD8-positive tumor infiltrating T cells), increasein immune cells that circulates in blood (total populations or specificsub-populations of lymphocytes, NK cells, monocytes, dendritic cells,macrophages, B cells, etc.), and/or presenting some differentialexpression pre-versus post-treatment only in either responding ornon-responding patients (as determined by RNA sequencing, mass flowcytometry, and/or other mass sequencing approach). Alternatively oradditionally, in some embodiments, such identification,characterization, and/or validation may involve the follow-up atmolecular level by screening the mRNA and/or protein expression of oneor more specific proteins or sets of proteins. In some embodiments, oneor more such techniques may allow identification or relevant informationfor evaluating the response to a CD38 Modulating Antibody Agent asdescribed herein, for example that may be is related to tissuedistribution and/or markers for specific cell populations within (ornearby) the tumor and/or circulating in blood.

Such approaches and immune-biological data may allow determination notonly of one or more efficacy and/or safety parameters orcharacteristics, but in some embodiments can provide a rationale forchoosing a particular dose , route or dosing regimen, for example thatmay be utilized in one or more clinical trials for a given indication,alone and/or in combination with other drugs, standard-of-careprotocols, or immunotherapies that can provide further therapeuticbenefits. Thus, in a series of further embodiments of the invention, aCD38 Modulating Antibody Agent as described herein is used in a methodof treating a patient suffering from a disease (such as cancer) orpreventing a disease (such as cancer) after determining the combinedpresence (and/or absence) of expression at RNA and/or protein level forone or more genes in cells or tissues of the patient (such as a tumor, ablood sample, or a blood fraction), post- or pre-treatment with such aformulation. Such methods may allow therefore defining a one or morebiomarkers, or a more complex gene expression signature (or cellpopulation distribution) that is associated to the therapeuticallyeffective amount of a desirable CD38 Modulating Antibody Agent, thetherapeutically relevant biomarker(s) that predicts that a subject mayhave an anti-tumor or anti-infective response after the treatment with aCD38 Modulating Antibody Agent, or the therapeutically relevantbiomarker(s) that predicts that a subject may respond to the treatmentwith a compound after the treatment with a CD38 Modulating AntibodyAgent.

Alternatively or additionally, in some embodiments dosing andadministration for a particular CD38 Modulating Antibody Agent asdisclosed herein can be preliminarily established and/or later evaluatedin view of CD38 expression in human cancers and/or other human tissues,for example by gathering data about CD38 distribution in stromal and/orimmune subsets in various cancers, tissues, and/or patients. Such datacan be generated by using common technologies (such as flow cytometry,mass cytometry, immunohistochemistry or mRNA expression libraries)across common cancer types and/or tissues (central nervous system,Esophagus, Stomach, Liver, Colon, Rectum, Lung, Bladder, Heart, Kidney,Thyroid, Pancreas, Uterus, Skin, Breast, Ovary, Prostate and testis) foridentifying relationship between CD38 expression in various immune andnon-immune subpopulations and/or its relation with cell infiltratemeasures and/or cancer-relevant markers associated with sub-sets ofcancer cells or immune cells (such as Foxp3 and PD-1/PD-L1). CD38expression can be confined (or not) to the immune subset in tumor tissue(such as in NK cells and other effector or regulatory immune cells), andcorrelations between CD38 expression and immune checkpoint inhibitorscan be determined if being positive, thus suggesting appropriate uses ofCD38 Modulating Antibody Agents in combinations with compounds targetingsuch immune checkpoint inhibitors.

Articles of Manufacture and Kits; In some embodiments of the invention,a CD38 Modulating Antibody Agent as described herein is provided in aseparate article of manufacture. In some embodiments of the invention,an article of manufacture containing a CD38 Modulating Antibody Agent isprovided in or with a container with a label. Suitable containers mayinclude, for example, bottles, vials, syringes, and test tubes. In someembodiments, a container may be formed from any or a variety ofmaterials such as glass or plastic. In some embodiments, a containerholds a composition that is effective for treating a particular disease,disorder, or condition, or stage or type thereof. In some embodiments, acontainer may have a sterile access port (for example the container maybe an intravenous solution bag or a vial having a stopper pierceable bya hypodermic injection needle). For example, in some embodiments, acompositions comprising a CD38 Modulating Antibody Agent as describedherein is packaged in clear glass vials with a rubber stopper and analuminium seal. The label on, or associated with, the containerindicates that the composition is used for treating the condition ofchoice.

In some embodiments, an article of manufacture may further comprise aseparate container comprising a pharmaceutically acceptable buffer, suchas phosphate-buffered saline, Ringer's solution and dextrose solutionand/or may further include other materials desirable from a commercialand user standpoint, including other buffers, diluents, filters,needles, syringes, and package inserts with instructions for use. Forexample, in some embodiments an article of manufacture may allowproviding each or the agent in an intravenous formulation as a sterileaqueous solution containing a total of 2 mg, 5 mg, 10 mg, 20 mg, 50 mg,or more that are formulated, with appropriate diluents and buffers, at afinal concentration of 0.1 mg/ml, 1 mg/ml, 10 mg/ml, or at a higherconcentration.

In some embodiments, a CD38 Modulating Antibody Agent as describedherein can be provided within the kits-of-parts in the form oflyophilized is to be reconstituted with any appropriate aqueous solutionthat provided or not with the kits, or other types of dosage unit usingany compatible pharmaceutical carrier. One or more unit dosage forms ofa CD38 Modulating Antibody Agent may be provided in a pack or dispenserdevice. Such a pack or device may, for example, comprise metal orplastic foil, such as a blister pack. In order to use correctly suchkits-of-parts, it may further comprise buffers, diluents, filters,needles, syringes, and package inserts with instructions for use in thetreatment of cancer.

In some embodiments, instructions that are associated with an article ofmanufacture or the kits as described herein may be in the form of alabel, a leaflet, a publication, a recording, a diagram, or any othermeans that can be used to inform about the correct use and/or monitoringof the possible effects of the agents, formulations, and other materialsin the article of manufacture and/or in the kit. Instructions may beprovided together with the article of manufacture and/or in the kit.

EXAMPLES Example 1: Generation of Antibodies that Bind CD38 in Vitro

Materials & Methods

CD38 antigen preparation. Recombinant, Histidine-tagged extracellulardomain of human, Cynomolgus monkey (Cyno), and murine CD38 proteins werepurchased from Sino Biological Inc. Protein reagent biotinylation wasdone using the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Scientific,Cat #21425). The CD38 antigen was concentrated to ˜1 mg/mL and bufferexchanged into PBS before addition of 1:7.5 molar ratio biotinylationreagents (EZ-Link Sulfo-NHS-Biotinylation Kit, Thermo Scientific, Cat#21425). The mixture was held at 4° C. overnight prior to another bufferexchange to remove free biotin in the solution. Biotinylation wasconfirmed through Streptavidin sensor binding of the labelled proteins.

Library interrogation and selection methodology for isolation ofanti-CD38 antibodies. Eight naïve human synthetic yeast libraries eachof ˜10⁹ diversity were designed, generated, and propagated forhigh-throughput screening and selection of yeast cell lines expressingmonoclonal antibodies as described previously (Xu Y et al, 2013;WO2009036379; WO2010105256; WO2012009568). Eight parallel selectionswere performed, using the eight naïve libraries for monomeric humanCD38-based selection.

For the first two rounds of selection, a magnetic bead sorting techniqueutilizing the Miltenyi MACs system was performed, essentially asdescribed (Siegel et al., 2004). Briefly, yeast cells (˜10¹⁰cells/library) were incubated with 3 ml of 100 nM biotinylated monomerichuman CD38 antigen for 15 minutes at room temperature in FACS washbuffer PBS with 0.1% BSA. After washing once with 50 ml ice-cold washbuffer, the cell pellet was re-suspended in 40 mL wash buffer, and 500μl Streptavidin MicroBeads (Miltenyi Biotec, Germany. Cat #130-048-101)were added to the yeast cells and incubated for 15 minutes at 4° C.Next, the yeast cells were pelleted, resuspended in 5 mL wash buffer,and loaded onto a MACS LS column (Miltenyi Biotec, Germany. Cat. No.130-042-401). After the 5 mL was loaded, the column was washed 3 timeswith 3 ml FACS wash buffer. Column was removed from magnetic field,yeast cells were eluted with 5 mL growth media, and then grownovernight.

Subsequent to the two MACS rounds, five rounds of sorting were performedusing flow cytometry (FACS). For the first round of FACS selection,approximately 4×10⁷ yeast cells were pelleted, washed three times withwash buffer, and incubated with 100 nM of each the biotinylatedmonomeric human, murine, and Cynomolgus CD38 antigen for 10 minutes atroom temperature. Yeast cells were then washed twice and stained withgoat anti-human F(ab′)2 kappa-FITC diluted 1:100 (Southern Biotech, USA;Cat. No. 2062-02) and either streptavidin-Alexa Fluor 633 (LifeTechnologies, USA; Cat. No. S21375) diluted 1:500, orExtravidin-phycoerthyrin (Sigma-Aldrich, USA; Cat. No. E4011) diluted1:50, secondary reagents for 15 minutes at 4° C. After washing twicewith ice-cold wash buffer, cell pellets were resuspended in 0.4 mL washbuffer and transferred to strainer-capped sort tubes. Sorting wasperformed using a FACS ARIA sorter (BD Biosciences) and sort gates weredetermined to select only CD38 binding. Murine- and Cyno-selectedpopulations from the first FACS round were combined into two pools.These pools were then sorted for human CD38 binding to identifycross-reactive binders in the second FACS round to decrease reagentpolyspecific binders (Xu Y et al., 2013). The fourth FACS roundconsisted predominantly of positive selection using 100 nM biotinylatedmonomeric CD38 as antigen. A sample of the selected clones were platedand sequenced.

Affinity Maturation of clones identified in naive selections. Heavychains from the fourth FACS sorting selection round outputs were used toprepare light chain diversification libraries used for four additionalselection rounds. The first selection round involved Miltenyi MACs beadsconjugated with either 100 nM biotinylated monomeric human CD38 asantigen or 200 nM biotinylated monomeric murine CD38 as antigen.Subsequent to the MACs bead selections, three rounds of FACS sortingwere performed. The first FACS round involved either human CD38 at 100nM or 10 nM or murine CD38 at 200 nM. In parallel to the second FACSround described above, competition selections were performed with 75-100nM of competitor IgG. After a selection round, a third positive sortwith human CD38 at 1 or 10 nM was done before plating. Individualcolonies from each FACS selection round were picked for sequencing IgG.

IgG and Fab production & purification. Yeast clones were grown tosaturation and then induced for 48 hrs at 30° C. with shaking. Afterinduction, yeast cells were pelleted and the supernatants were harvestedfor purification. IgGs were purified using a Protein A column and elutedwith acetic acid, pH 2.0. Fab fragments were generated by papaindigestion and purified over CaptureSelect IgG-CH1 affinity matrix (LifeTechnologies; Cat. No. 1943200250).

Affinity Measurements of anti-CD38 Antibodies The affinity for the CD38antibodies was determined by measuring their KD by Forte Bio. Forte Bioaffinity measurements were performed by loading IgGs on-line onto AHQsensors as described (Estep P et al., 2013). Briefly, sensors wereequilibrated off-line in assay buffer for 30 minutes and then monitoredon-line for 60 seconds for baseline establishment. For avid bindingmeasurement, sensors with loaded IgGs were exposed to 200 nM of human,cynomolgus, or murine CD38 for 3 minutes, afterwards they weretransferred to assay buffer for 3 minutes for off-rate measurement.Monovalent binding measurements were obtained by loading biotinylatedCD38 monomer on SA sensors followed by exposure to 200 nM antibody.Kinetics data were fit using a 1:1 binding model of data analysissoftware provided by Forte Bio. The Kd values that were established inthis assay for the reference agonistic anti-CD38 antibodies are thefollowing: for IB4, 0.9×10⁻⁸ M for human CD38 and no binding tocynomolgus CD38, for IB4

Avidity binding measurements of anti-CD38 Antibodies: Ni-NTA sensorswere equilibrated off-line in assay buffer for 30 min and then monitoredon-line for 60 seconds for baseline establishment. They were loaded with4.2 nM antigen (recombinant human CD38 HIS tagged) for 50 min,afterwards they were transferred to assay buffer for 0.5 min for washand again for 1 min in assay buffer for base line determination. Thenthe antibody was associated at different concentrations (as described inFIGS. 14 and 12) for 30-50 min. Afterwards they were transferred toassay buffer for 20-30 min for off-rate measurement. Kinetics data werefit using a 1:1 binding model in the data analysis software provided byForteBio.

Alternatively affinity for the anti-human CD38 antibodies was determinedby measuring their K_(D) by SPR in a Biacore 2000 using a CM-5 Sensorchip with an ambient experiment temperature of 25° C. Anti-humanantibody was initially immobilised across all flow cells in analysisbuffer (pH 7.4, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20) toan RU of between 12,000-14,000 over 10 minutes. The ligand (antibodytest articles) was sub sequentially loaded to a capture level between54-208RU. The analyte (recombinant human CD38 his tagged) was thenassociated in analysis buffer from a 2-fold dilution starting at 3200 nMwith a lowest concentration of 0.78 nM for 6 minutes. Dissociation wasperformed in analysis buffer over 10 minutes. Regeneration steps betweensample concentrations were performed in 3 M MgCl₂, three times for 0.5minutes. A flow rate of 25 μl/min was maintained throughout the process.Kinetics data were fit using a global fit on the analysis softwareprovided by Biacore with reference subtraction.

Epitope Binning: Epitope binning of the antibodies was performed on aForte Bio Octet Red384 system (Pall Forte Bio Corp., USA) using astandard sandwich binning assay. The anti-human CD38 antibody was loadedonto AHQ sensors and unoccupied Fc-binding sites on the sensor wereblocked with a non-relevant human IgG1 antibody. Sensors were exposed to100 nM target antigen followed by a second anti-CD38 antibody, thereference monoclonal agonistic mouse anti-human CD38 antibodies (IB4,kindly provided by Prof. F. Malavasi at Univ. Torino, Italy). Data wasprocessed using Forte Bio Data Analysis Software 7.0. Additional bindingby second antibody after antigen association indicates an unoccupiedepitope, while no binding indicates epitope blocking.

Binding of anti-CD38 Antibodies to CD38-expressing cells: The candidatehits are evaluated by analysing the binding to human PBMC. To this aim,PBMCs were prepared from whole blood from 3 human donors and incubatedfor 30 minutes with aCD38-b-329 or DARA at final concentrations of 1 μM,200 nM, 40 nM, 8 nM, 1.6 nM, 320 pM, 64 pM, 13 pM and 2.5 pM. Cells werethen washed and labelled with an AF488 secondary antibody. Cells werethen incubated with additional surface staining antibodies: anti-CD3PE-Cy7, anti-CD4 APC and anti-CD8 BV451. Sample acquisition wasperformed using 8-colour (three laser) BD FACSCanto II cytometer,running on the BD FACSDiva software (BD Biosciences). Post-analysisprocessing and was conducted using FCS Express (v3.0) software (DeNovosoftware). The relative proportions (%) of the different cellpopulations and Median Fluorescence Intensity (MFI) data were reportedto 2 decimal places.

Recloning, producing, and characterizing of aCD38-b-329 as human IgG1expressed in mammalian cells. Synthesis of codon optimized VH and VLcoding sequences for the antibody was performed by Genewiz. cDNAs ofvariable regions were cloned into the antibody expression vector(Icosagen, EST) containing human IgG1 heavy chain and kappa light chainconstant regions (P01857 and P01834 respectively). Full length heavy andlight chain cDNAs were verified by sequencing in final vectors and thenrecloned for expressing them using the QMCF Technology (Icosagen) astable episomal expression system that uses CHO-based cells(CHOEBNALT85) and appropriate vectors for production of recombinantproteins, antibodies, CHOEBNALT85 cells were transfected with 1 μg ofthe expression plasmids for antibody production. 48 h after thetransfection 700 μg/ml of G418 was added to select plasmid containingcell population. For the production, temperature was shifted to 30° C.and the cultures were additionally fed. At the end of the production theculture supernatants were clarified by centrifugation (1000 g, 30minutes, and 15° C.), PMSF was added and supernatants were processed orfrozen until purification. hIgG1 antibodies were purified by MabSelectSuRe affinity chromatography followed by Superdex 200 gel filtrationinto either 25 mM NaOAc pH 5,5; 50 mM NaCl or PBS. Human IgG1 antibodiesproduced in CHOEBNALT85 cells were characterized for affinity towardsrecombinant human CD38, cross reactivity towards murine, rat, rabbit andcynomolgus CD38 and epitope binning versus the selected CD38 bindingantibodies using recombinant rabbit CD38 (65003-T08H-20; SinoBiological) and recombinant rat CD38 (80229-R08H-2; Sino Biological).

aCD38-b-329 epitope mapping Different sets of linear, single loop,β-turn mimics, disulfide bridge mimics, discontinuous disulfide bridges,discontinuous epitope mimics peptides representing the human CD38sequence (Uniprot record no. P28907) were synthesized using solid-phaseFmoc synthesis (Pepscan B V, The Netherlands; Timmermann P et al., 2007;Langedijk J P et al., 2011). The binding of antibody to each of thesynthesized peptides was tested in a pepscan-based ELISA (Pepscan, TheNetherlands). The peptide arrays were incubated with primary antibodysolution (overnight at 4° C.). After washing, the peptide arrays wereincubated with a 1/1000 dilution of an appropriate antibody peroxidaseconjugate (2010-05; Southern Biotech) for one hour at 25° C. Afterwashing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazolinesulfonate (ABTS) and 20 μl/ml of 3 percent H₂O₂ were added. After onehour, the color development was measured.

The color development was quantified with a charge coupled device(CCD)—camera and an image processing system. The values obtained fromthe CCD camera range from 0 to 3000 mAU, similar to a standard 96-wellplate ELISA-reader. To verify the quality of the synthesized peptides, aseparate set of positive and negative control peptides was synthesizedin parallel and screened with irrelevant, control antibodies.

Anti-human CD38 Ab Competition Assays: Antibody competitions wereperformed on a Forte Bio Octet Red96 system (Pall Forte Bio Corp., USA)using a standard sequential binding assay. 0.625 μg/mL of recombinanthuman CD38 his tagged was loaded onto Ni-NTA Biosensors for 300 s. Afterwash for 15 s and a base line step for 60 s on kinetic buffer sensorswere exposed to 66.6 nM of first antibody (Daratumumab) for 600 sfollowed by a second anti-CD38 antibody (Daratumumab (control) oraCD38-b-329) (also at 66.6 nM for 600 s). Data was processed using ForteBio Data Analysis Software 9.0. Additional binding by a second antibodyindicates an unoccupied epitope (no competition for the epitope), whileno binding indicates epitope blocking (competition) for the epitope.

Results

Monoclonal antibodies (mAb) binding to recombinant human CD38extracellular protein sequence (rhCD38) have been isolated using ayeast-based antibody presentation library as described in the Materials& Methods. These antibodies were sequenced and unique clones wereproduced in yeast cells (Barnard G C et al., 2010). The cell culturesupernatants for each yeast clone expressing a unique antibody sequencewas screened for rhCD38 binding.

The KD values (for affinity and avidity measurements) and crossingbinning analysis for selected antibodies are provided in Table 1:

TABLE 1 Affinity Avidity Epitope K_(D) Human K_(D) Human K_(D) HumanCross- CD38-HIS CD38-HIS CD38-HIS binning Monovalent MonovalentMonovalent Antibody group (M) (Octet) Isotype (M) (Biacore) (M) IsotypeaCD38-b-329 B 3.38E−09 IgG1 0.62E−09 — — Daratumumab F 8.28E−08 IgG1 —1.80E−10 IgG1

Binding of anti-CD38 antibodies to recombinant monovalent human CD38measured by Octet and Biacore and compared to Daratumumab, is shown inFIGS. 11A, 12 and 14.

Based on the binding to rhCD38, sequence uniqueness and expressionlevels a panel of mAbs was identified. These antibodies were furthercharacterized for binding to recombinant Cynomolgus monkey and mouseCD38 extracellular domain protein sequences. In addition, epitopebinning was performed to determine whether the identified antibodiesbind to epitopes overlapping with those of the reference agonisticanti-CD38 antibody IB4 (Ausiello C M et al., 2000; Ferrero E et al.,2004). The clones were characterized presenting IgG binding values tomonovalent rhCD38 and/or recombinant cynomolgus CD38 extracellularprotein sequences that is comprised between 10⁻⁸ M and 10⁻¹⁰ M. Inaddition, each antibody was characterized as competing or not withreference agonistic anti-CD38 antibody IB4 (or commercially availableDaratumumab, DARA). The selected antibodies belong to different crossbinning groups, as shown on Table 1. The antibody clones were alsoevaluated at the level of binding to human cells strongly expressingCD38, such as lymphoblast-like, Raji cells by flow cytometry, usingCHO-S cells as negative control. Further, binding to human PBMC (FIG. 3)was confirmed.

Finally, in order to eliminate antibody sequences that would be prone toaggregation and non-specific interaction, the antibodies were screenedin a Poly Specific Reagent (PSR) assay and Affinity-CaptureSelf-Interaction Nanoparticle Spectroscopy (AC-SINS), an approach thatallows high-throughput screening for early-stage antibody development(Liu Y et al., 2014). None of the antibodies scored positive in thelatter assays and as such were not removed from the panel.

Among the selected hits that were sequenced and characterized asdescribed above, the clone aCD38-b-329 is an antibody presenting novelcomplementarity determining regions (CDRs; FIG. 2) that competes forhuman CD38 binding neither with IB4 nor with Daratumumab. Indeed, theepitope mapping study that has been performed using Pepscan technologywould indicate that aCD38-b-329 binds human CD38 in a region that is notonly distinct from the one published for Daratumumab (two beta-strandscontaining amino acids 233-246 and 267-280; FIG. 2B) or the ones thatare reported for other anti-human CD38 antibodies (see table 2 inWO2015123687) and binds human CD38 extracellular protein sequences witha Kd value in the 10⁻⁹ M range. The antibody competition assay showedthat Daratumumab does not compete with aCD38-b-329 for the same epitope(FIG. 13).

Thus, the aCD38-b-329 sequences (FIG. 2A) identify antibodies thatspecifically bind CD38, and whose agonistic activities associated to thefunctional features defining CD38 Modulating Antibody Agents, as thatterm is used herein, can be functionally evaluated by cell-based assaysor animal models. The analysis of aCD38-b-329-LCDR3 shows the presenceof a DG (Asp-Gly) motif which represents a potential target formodification during manufacturing that may affect some of the antibodyproperties. At this scope, aCD38-b-329-based antibody libraries in whicheither one or both residues are substituted and, among all such mutants,only a limited number of them maintained the binding to human CD38, forexample as expressed on the surface of Daudi cells (see Example 2). Inthis manner, the corresponding aCD38-b-329-based antibody variants canbe tested as maintaining full properties of CD38 Modulating AntibodyAgents, or simply having CD38 binding properties. Further validation ofthese variants may be pursued by using the assays disclosed in theExamples.

Example 2: Cell-Based Models for Validating CD38 Modulating AntibodyAgents

Materials & Methods

In vitro T cell activation assay: Previously frozen primary human pan Tcells (Stemcell Technologies) were labelled with eFluor450 fluorescentdye (Life Technologies) and 0.15×10⁶ cells per well were incubated for72hrs in 96-well plates pre-coated with anti-CD3 antibody (0.1 μg/mlcoating concentration, clone OKT3, eBiosciences) and anti-CD38modulating antibodies coated at concentrations of 10, 5 and 2.5 μg/ml inRPMI 1640 (Life Technologies) containing 10% FBS (Sigma), 2 mML-Glutamine (Life Technologies) and 10,000 U/ml Pen-Strep (Sigma).Readout of T cell proliferation was done by acquisition on the flowcytometer, excluding dead cells labelled with a viability dye (ZombieNIR, BioLegend) and discriminating surface markers by staining withfluorochrome labelled antibodies (CD8-FITC clone HIT8a eBiosciences,CD25-PE clone M-A251 Biolegend, CD4-BV510 clone RPA-T4 BioLegend,CD38-PE-Cy7 clone HB_7, eBiosciences, CD137-APC clone 4B4-1 BioLegend).Cytokine analysis in supernatants was conducted using the Meso ScaleDiscovery MSD platform, determining the expression of IFNg, IL-2, IL-10,TNFa, and GM-CSF according to the manufacturer's instructions (Multiplexassay kits, Meso Scale Discovery; asterisk in figure indicates valuesabove fit curve range).

In vitro ADCC assay: Antibody-dependent cell-mediated cytotoxicityassays (ADCC assays) were performed for the characterization ofanti-human CD38 antibodies using Daudi (CD38 positive) human cell lineas a target cell with human PBMC as the source of effector cells.Effector to Target ratios would be evaluated at 50 to 1 or 25 to 1 withtest articles (anti-CD38 primary antibodies or Rituximab as a control)to be evaluated with top concentration of 10 μg/ml followed by a logseries (7 points) in triplicate for 4 hours at 37° C. 5% CO2. PBMCs wereprimed with IL-2 and IL-2 was present during co-culture assays. Prior toin-vitro culture, target cell lines were labelled with 1 μM Calcein AMand incubated with 2.5 mM probenecid. Lysed cells release the loadedCalcein into the supernatant, which allows for fluorescent measurement.Calcein AM release was analysed by excel and GraphPad software analysisto generate dose response curves by normalization where 1% saponintreatment values will be used to determine maximal lysis. Percentagetarget cell lysis was plotted on an XY chart, graphing normalizedCalcein AM percentage release against the log of the concentration, andthe data fit to a no-linear regression curve from which the EC50 wascalculated.

In vitro CDC assay: CDC activity to CD38 expressing human cell lines(Daudi) was examined by treating cells with test articles (anti-CD38primary antibodies or Rituximab as control) at a top concentration of 10μg/ml followed by a log dilution series (7 points) in triplicate with afinal concentration of 10% normal human serum complement. Samples werecultured for 3 hours at 37° C. 5% CO2. Following culture conditions,cells washed and re-suspended in 1×PBS with propidium iodide (PI) at afinal concentration of 5 μg/ml prior to flow cytometry analysis. Totalcells were examined by flow cytometry during sample acquisition.Percentage of PI positive cells were plotted on an XY chart, graphingpercentage PI against the log of the concentration, and the data fit toa non-linear regression curve from which the EC50 is calculated.

Direct cell death assay. Direct proapoptotic activity to CD38 expressinghuman cell lines (Daudi) was examined by treating cells with testarticles (anti-CD38 primary antibodies) or Rituximab as a control at atop concentration of 10 μg/ml followed by a log dilution series (7points) in triplicate. Cell death by Fcγ receptor-mediated cross-linkingactivity was examined by treating cells with test articles (anti-CD38primary antibodies or Rituximab) as a control at a top concentration of10 μg/ml followed by a log serial dilution (7 points) in triplicatefollowed by 5 μg/ml rabbit anti-human Fcγ F(ab′)2 (secondary antibody).Samples were cultured for 24 hours at 37° C. 5% CO2. Following cultureconditions, cells were washed and resuspended in Annexin V bindingbuffer and 7-AAD to examine cell death by flow cytometry analysis. Totalcells were examined by flow cytometry during sample acquisition.Percentage of late apoptotic cells were plotted on an XY chart, graphingpercentage Annexin V-positive and 7-AAD-positive cells against the logof the concentration and the data fit to a non-linear regression fromwhich the EC50 is calculated.

Statistics. Prism software (GraphPad) was used to perform curve fittingand to determine EC50 values and maximal activity.

Results

The EC50 values and percentage lysis results from the ADCC and CDCassays are shown in Tables 2 and 3, compared to the results fordaratumumab in the same experiment:

TABLE 2 ADCC data for Target Daudi cells: Exper- EC50 Max. EC50 ug/mlMax. Lysis Antibody iment ug/ml lysis % DARA DARA aCD38-b-329 1 0.011 500.004 36 aCD38-b-329 2 0.0062 49 0.0025 58

TABLE 3 CDC data - 10% complement, target Daudi cells: Exper- EC50 Max.EC50 ug/ml Max. Lysis Antibody iment ug/ml lysis % DARA DARA aCD38-b-3299 NA NA 0.11 92

The aCD38-b-329 candidate antibody, as other antibodies that have beencharacterized in Example 1, has been further evaluated with respect toimmune cells. In a first series of experiments, aCD38-b-329 shows dosedependent binding to human T cells (FIG. 3). When tested using T cells,for instance when aCD38-b-329 is used for coating a plate for culturingsuch cells, aCD38-b-329 strongly increases human T cell activation whilethe reference anti-CD38 antibody (DARA) is displaying much weakeragonist activity (FIG. 4A). The agonist activity of aCD38-b-329 isfurther emphasized by stronger proinflammatory cytokine secretion byhuman T cells triggered by aCD38-b-329 when compared to DARA (FIG. 4B).In addition, aCD38-b-329 and DARA show comparable activity in ADCCassays, but only the latter one present CDC activity (FIG. 5A). Thereduced CDC effect triggered by aCD38-b-329 would provide an anti-CD38antibody having increased safety due to reduced infusion site reaction.DARA and aCD38-b-329 show also comparable activity in killing of CD38expressing tumour cells (Daudi) by human PBMC, even though only theactivity of aCD38-b-329 appears enhanced by antibody cross-linking (FIG.5B).

In conclusion, aCD38-b-329 has been characterized as an exemplaryanti-CD38 antibody that presents the activities of a CD38 ModulatingAntibody Agent with respect to immune cells in different experimentalset ups.

Example 3: Validation of CD38 Modulating Antibody Agent in Animal Models

Materials & Methods

Lymphoma cells-based models Daudi and Ramos human Burkitt Lymphoma cellswere cultured in RPMI 1640 containing 2 mM L-glutamine supplemented with10% fetal bovine serum+1 mM Na Pyruvate+4.5 g/L Glucose+10 mM Hepes.Healthy female cb17 SCID mice were obtained from Charles River. Tumorswere induced by intravenous injection of 5×10⁵ Daudi cells or 10⁶ Ramoscells in 200 μL of RPMI 1640 into the caudal vein of the animals. Cellinjection was performed 24 to 72 hours after a whole body irradiationwith a γ-source (1.44 Gy/mouse, 60Co, BioMep, Bretenières, France). Micewere randomized into treatment groups by bodyweight, 8 mice per group.Animals from group 1 received intravenous injections of vehicle at 5ml/kg twice a week for three consecutive weeks (TW×3). Animals fromgroup 2 received intravenous injections of DARA at 10 mg/kg/inj. twice aweek for three consecutive weeks (TW×3). Animals from group 3 receivedintravenous injections of aCD38-b-329 at 10 mg/kg/inj. twice a week forthree consecutive weeks (TW×3). Mice were sacrificed after a maximum of8 weeks.

Results

The therapeutic properties of aCD38-b-329 can be tested in animal modelfor human cancer, in particular using immunocompromised mice where theproperties of a CD38 Modulating Antibody Agent with respect to thekilling of human tumour cells can be more appropriately evaluated.aCD38-b-329 shows remarkable potency to increase survival of miceintravenously injected with two different types of human lymphoma cells,an effect that is superior to Daratumumab (DARA) in the Daudi model(FIG. 6A), and superior to the control group in both models (FIG. 6A andB).

These properties, not only in terms of animal survival but also withconcurrent immunological effects can be further investigated in other invivo models for human tumors (in particular solid cancers) that arebased on the injection with either human cancer lines or human primarycancer cells, in which solid tumors grow subcutaneously, as described inthe literature (Morton J J et al. 2016; Holzapfel B M et al., 2015), andin ex vivo models based on the use of tumour samples directly isolatedfrom patients from which tumour cells and immune cells are isolated andtested in vitro for their response to the anti-CD38 antibodies, asmeasured by cell activation, proliferation, cytokine production and/orcell death. Additional features such as abscopal effects or changes ingene expression in selected tissues or biological materials can beevaluated, possibly by administering aCD38-b-329 in different dosesand/or in combination with other anti-cancer agents (such as inhibitorsof kinases or of other enzymes, antibodies, radio/chemo-therapy,adjuvants, or vaccines).

Example 4: Generation of Variants of aCD38-b-329

To prevent aspartate isomerisation, a library of potential substitutionsfor the VL CDR3 DG sequence was generated. Two yeast display librarieswere generated for aCD38-b-329 in order to remove a DG motif. The firstlibrary was based on degenerate primers NNKNNK around both the aspartateand the glycine. This library had a diversity of 400. The second librarywas based on a degenerate primer NNK focused on the aspartate whilepreserving the glycine, with a diversity of 20. These libraries weresorted in a single round on human CD38 monomer at 10 nM and 96 from eachlineage were sequenced, produced, and characterized as above.Additionally, a total of five 96-well plates were picked from the NNKNNKlibraries to screen for binding to rhCD38 in Octet. A limited number ofsubstitutions were tolerated and the variants showing the bestaffinities were selected and the variants tested for binding the rhCD3in Octet. Variants showing the best affinities were selected formammalian production and further characterisation using the assays asdescribed above in Example 2.

Functional characterization of variants: Lymphoma cells-based modelsRaji and Ramos human Burkitt Lymphoma cells were cultured in RPMI 1640containing 2 mM L-glutamine supplemented with 10% fetal bovine serum+1mM Na Pyruvate+4.5 g/L Glucose+10 mM Hepes. Healthy female cb17 SCIDmice were obtained from Charles River. Tumors were induced byintravenous injection of 5×10⁵ Raji cells or 10⁶ Ramos cells in 200 μLof RPMI 1640 into the caudal vein of the animals. Cell injection wasperformed 24 to 72 hours after a whole body irradiation with a γ-source(1.44 Gy/mouse, 60Co, BioMep, Bretenières, France). Mice were randomizedinto treatment groups by bodyweight, 10 mice per group. Animals fromgroup 1 received intravenous injections of vehicle at 5 ml/kg twice aweek for three consecutive weeks (TW×3). Animals from group 2 receivedintravenous injections of DARA at 10 mg/kg/inj. twice a week for threeconsecutive weeks (TW×3). Animals from group 3 received intravenousinjections of aCD38-b-329 at 10 mg/kg/inj. twice a week for threeconsecutive weeks (TW×3). Mice from group 4 received intravenousinjections of aCD38-b-329-m6 at 10 mg/kg/inj. twice a week for threeconsecutive weeks (TW×3), and mice from group 5 received intravenousinjections of DaCD38-b-329-m7 at 10 mg/kg/inj. twice a week for threeconsecutive weeks (TW×3) Mice were sacrificed at the time indicated inthe figure.

Results

The CDR1-FR2-CDR2-FR3-CDR3 sequence of the VH and VL chains of thevariant antibodies are shown in FIG. 7 (SEQ ID NO: 4 and SEQ ID NO: 16for aCD38-b-329-m6, and SEQ ID NO: 4 and SEQ ID NO: 17 foraCD38-b-329-m7). Daudi cell binding experiments confirmed that bindingof the variant antibodies to CD38 was comparable with the parentalclones and with daratumumab, Table 4 (FIG. 8 and FIG. 11).

TABLE 4 Biacore LCDR3 Octet Affinity Affinity EC50 Sequence K_(D) (M)K_(D) (M) ug/ml Max MFI aCD38-b-329 QQDGAVFT 2.38E−09 0.62E−09 0.7342508 (SEQ ID NO: 7) aCD38-b-329-m6 QQDEAVFT 1.99E−08 4.70E−09 0.651 2297(SEQ ID NO: 10) aCD38-b-329-m7 QQDSAVFT 2.60E−08 4.90E−09 0.745 2213(SEQ ID NO: 11) Daratumumab — — — 0.945 2495

The variants showed comparable ADCC activity to the parental clones anddaratumumab (FIG. 9). The variants had a lower EC50ug/ml for ADCCactivity than the parental strain but comparable maximum lysis (Table5).

TABLE 5 ADCC target lysis: EC50 Max. % ug/ml lysis aCD38-b-329 0.0062 49aCD38-b-329-m6 0.0024 47 aCD38-b-329-m7 0.0035 43 Daratumumab 0.0025 58IgG1 Isotype NA 0

Peptide mapping samples were prepared through DTT reduction andiodoacetamide alkylation, digested using trypsin and analyzed on aLC-UV-MS system. The level of isomerized and non-isomerized peptideswere determined and % of isomerized peptide are shown in Table 6:

TABLE 6 Antibody % isoD aCD38-b-329 7.4% aCD38-b-329-m6 0.0%aCD38-b-329-m7 0.0%

The therapeutic properties of aCD38-b-329 and its variantsaCD38-b-329-m6 and aCD38-b-329-m7 were tested in animal models for humancancer, in particular using immunocompromised mice where the propertiesof a CD38 Modulating Antibody Agent with respect to the killing of humantumour cells can be more appropriately evaluated. aCD38-b-329 variantsaCD38-b-329-m6 and aCD38-b-329-m7 show remarkable potency to increasesurvival of mice intravenously injected with two different types ofhuman lymphoma cells, an effect that is superior to Daratumumab (DARA)both models (Raji and Ramos) (FIG. 10).

Example 5: Antibody Binding to Mutant CD38

Materials and Methods: Two mutant version of human CD38 wereconstructed. In one version D was mutated to G at position 202 (D202G)and in the second version S was mutated to F at position 274 (S274F).

The binding of aCD38-b329 to each of the mutated CD38 proteins wasassessed, and compared to daratumumab.

Results

The results showed that binding of aCD38-b-329 was not affected by theintroduction of mutation D202G or mutation S274F into human CD38. Thiscompares to Daratumumab where antibody binding was affected by theintroduction of mutation S274F, but was not affected by the introductionof mutation D202G into human CD38. These results confirm thataCD38-b-329 binds to a different epitope than daratumumab.

A summary of the sequences included in the application is provided below

SEQ ID NO Description of Antibody sequences Also referred to as: 1aCD38-b-329 variable heavy chain CDR1 aCD38-b-329-HCDR1 2 aCD38-b-329variable heavy chain CDR2 aCD38-b-329-HCDR2 3 aCD38-b-329 variable heavychain CDR3 aCD38-b-329-HCDR3 4 aCD38-b-329 variable heavy chain CDR 1,aCD38-b-329-HCDR123 2, 3 and FR 2, 3 aCD38-b-329-m6-HCDR123aCD38-b-329-m7-HCDR123 5 aCD38-b-329 variable light chain CDR1aCD38-b-329-LCDR1 6 aCD38-b-329 variable light chain CDR2aCD38-b-329-LCDR2 7 aCD38-b-329 variable light chain CDR3aCD38-b-329-LCDR3 8 aCD38-b-329 variable light chain CDR 1,aCD38-b-329-LCDR123 2, 3 and FR 2, 3 9 Human CD38 Uniprot sequenceP28907 10 aCD38-b-329-m6 variable light chain CDR3 aCD38-b-329-m6 -LCDR3 11 aCD38-b-329-m7 variable light chain CDR3 aCD38-b-329-m7 - LCDR312 aCD38-b-329 variable heavy chain CDR 1, aCD38-b-329-VH 2, 3, and FR1, 2, 3, 4, aCD38-b-329-m6-VH aCD38-b-329-m7-VH 13 aCD38-b-329-m6variable light chain CDR 1, aCD38-b-329-m6-VL 2, 3, and FR 1, 2, 3, 4,14 aCD38-b-329-m7 variable light chain CDR 1, aCD38-b-329-m7-VL 2, 3,and FR 1, 2, 3, 4, 15 aCD38-b-329 variable light chain CDR 1,aCD38-b-329-VL 2, 3, and FR 1, 2, 3, 4, 16 aCD38-b-329-m6 variable lightchain CDR 1, aCD38-b-329-m6-LCDR123 2, 3 and FR 2, 3 17 aCD38-b-329m7-variable light chain CDR 1, aCD38-b-329-m7-LCDR123 2, 3 and FR 2, 318 Daratumumab variable heavy chain 19 Daratumumab variable light chain

EQUIVALENTS AND SCOPE

Those skilled in the art will appreciate that the present invention isdefined by the appended claims and not by the Examples or otherdescription of certain embodiments included herein.

Similarly, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise.

Unless defined otherwise above, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, genetics and protein andnucleic acid chemistry described herein are those well known andcommonly used in the art, or according to manufacturer's specifications.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited.

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1. An antibody or antigen-binding fragment thereof, comprising theaCD38-b-329-HCDR3 amino acid sequence as variable heavy chaincomplementarity determining region
 3. 2. The antibody or antigen-bindingfragment thereof of claim 1, further comprising a) aCD38-b-329-HCDR1amino acid sequence as variable heavy chain complementarity determiningregion 1; and b) aCD38-b-329-HCDR2 amino acid sequence as variable heavychain complementarity determining region
 2. 3. The antibody orantigen-binding fragment thereof of claim 1 or claim 2, furthercomprising: a) aCD38-b-329-LCDR1 amino acid sequence as variable lightchain complementarity determining region 1; b) aCD38-b-329-LCDR2 aminoacid sequence as variable light chain complementarity determining region2; and c) aCD38-b-329-LCDR3 amino acid sequence as variable light chaincomplementarity determining region
 3. 4. The antibody or antigen-bindingfragment thereof of claim 1, 2 or 3, wherein the antibody orantigen-binding fragment thereof comprises a variable heavy chaincomprising aCD38-b-329-HCDR123 amino acid sequence.
 5. The antibody orantigen-binding fragment thereof according to anyone of claims 1 to 4,wherein the antibody or antigen-binding fragment thereof furthercomprises a variable light chain comprising aCD38-b-329-LCDR123 aminoacid.
 6. The antibody or antigen-binding fragment thereof according toany one of claims 3 to 5, wherein the antibody or antigen-bindingfragment thereof is mutated to remove the DG motif in the LCDR3 region.7. The antibody or antigen-binding fragment thereof according to claim6, wherein the antibody or antigen-binding fragment thereof comprises anLCDR3 region selected from the group consisting of QQDEAVFT andQQDSAVFT.
 8. The antibody or antigen-binding fragment thereof accordingto any one of claims 1 to 7 wherein the antibody or an antigen-bindingfragment thereof is selected from the group consisting of: a) anantibody or antigen binding fragment thereof comprising an HCDR1comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising thesequence of SEQ ID NO: 2, an HCDR3 comprising the sequence of SEQ ID NO3, an LCDR1 comprising the sequence of SEQ ID NO: 5, an LCDR2 comprisingthe sequence of SEQ ID NO: 6, and an LCDR3 comprising the sequence ofSEQ ID NO: 7; b) an antibody or antigen binding fragment thereofcomprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2comprising the sequence of SEQ ID NO: 2, an HCDR3 comprising thesequence of SEQ ID NO 3, an LCDR1 comprising the sequence of SEQ ID NO:5, an LCDR2 comprising the sequence of SEQ ID NO: 6, and an LCDR3comprising the sequence of SEQ ID NO: 10; c) an antibody or antigenbinding fragment thereof comprising an HCDR1 comprising the sequence ofSEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 2, an HCDR3comprising the sequence of SEQ ID NO 3, an LCDR1 comprising the sequenceof SEQ ID NO: 5, an LCDR2 comprising the sequence of SEQ ID NO: 6, andan LCDR3 comprising the sequence of SEQ ID NO: 11; d) an antibody orantigen binding fragment thereof comprising a heavy chain variableregion comprising the sequence of SEQ ID NO: 4 and a light chainvariable region comprising the sequence of SEQ ID NO: 8; e) an antibodyor antigen binding fragment thereof comprising a heavy chain variableregion comprising the sequence of SEQ ID NO: 4 and a light chainvariable region comprising the sequence of SEQ ID NO: 16; f) an antibodyor antigen binding fragment thereof comprising a heavy chain variableregion comprising the sequence of SEQ ID NO: 4 and a light chainvariable region comprising the sequence of SEQ ID NO: 17; g) an antibodyor antigen binding fragment thereof comprising a heavy chain variableregion comprising the sequence of SEQ ID NO: 12 and a light chainvariable region comprising the sequence of SEQ ID NO: 15; h) an antibodyor antigen binding fragment thereof comprising a heavy chain variableregion comprising the sequence of SEQ ID NO: 12 and a light chainvariable region comprising the sequence of SEQ ID NO: 13; and i) anantibody or antigen binding fragment thereof comprising a heavy chainvariable region comprising the sequence of SEQ ID NO: 12 and a lightchain variable region comprising the sequence of SEQ ID NO:
 14. 9. Theantibody or an antigen-binding fragment thereof of any preceding claimwherein the antibody or antigen-binding fragment thereof isa-fucosylated.
 10. An antibody or antigen-binding fragment thatspecifically binds to an epitope of human CD38, wherein the epitopecomprises one or more amino acid residues comprised in amino acids 65-79of SEQ ID NO:
 9. 11. The antibody or antigen or antigen-binding fragmentof claim 9 wherein the epitope comprises amino acids 65-79 of SEQ ID NO:9.
 12. An affinity matured variant of an antibody or antigen bindingfragment thereof of any one of claims 1 to
 9. 13. The antibody orantigen-binding fragment thereof according to any one of claims 1 to 12,wherein the antibody or antigen-binding fragment thereof is a monoclonalantibody, a domain antibody, a single chain antibody, a Fab fragment, aF(ab′)2 fragment, a single chain variable fragment (scFv), a scFv-Fcfragment, a single chain antibody (scAb), an aptamer, or a nanobody. 14.The antibody or antigen-binding fragment thereof according to any one ofclaims 1 to 13, wherein the antibody or antigen-binding fragment thereofis a rabbit, mouse, chimeric, humanized or fully human antigen-bindingantibody.
 15. The antibody or antigen-binding fragment thereof accordingto any one of claims 1 to 13, wherein the antibody is selected from thegroup consisting of IgG1, IgG2, IgG3, and IgG4 isotype antibodies. 16.The antibody or antigen-binding fragment thereof according to any one ofclaims 1 to 15, wherein the antibody or antigen-binding fragment thereofis comprised in a bispecific antibody, a multispecific antibody, or animmunoconjugate further comprising a therapeutic or diagnostic agent.17. The antibody or antigen-binding fragment thereof according to anyone of claims 1 to 16, wherein the antibody or antigen-binding fragmentthereof binds the extracellular domain of human CD38.
 18. The antibodyor antigen-binding fragment thereof according to any one of claims 1 to17, wherein the antibody or antigen-binding fragment thereof binds cellsexpressing human CD38 on their surface and is a CD38 Modulating AntibodyAgent.
 19. A nucleic acid molecule encoding the antibody orantigen-binding fragment thereof of any of the claims 1 to
 18. 20. Anucleic acid vector comprising the nucleic acid molecule of claim 19.21. A host cell comprising the nucleic acid vector of claim
 20. 22. Amethod for producing an antibody or antigen-binding fragment thereofaccording to any one of claims 1 to 18 by culturing a host cell of claim21.
 23. A composition comprising an antibody or antigen-binding fragmentthereof according to any one of claims 1 to
 18. 24. The composition ofclaim 23 that further comprises a pharmaceutically acceptable carrier orexcipient.
 25. The pharmaceutical composition of claim 24, wherein saidcomposition is for use in the treatment of cancer.
 26. Use of anantibody or antigen-binding fragment thereof according to any one ofclaims 1 to 18, or of a composition of claim 23 in the manufacture of amedicament for treating a cancer.
 27. Use of an antibody orantigen-binding fragment thereof that competes with an antibodyaccording to anyone of claims 1 to 18 for the binding of CD38 in themanufacture of a medicament for treating cancer.
 28. A method oftreating cancer in a subject, comprising administering to the subject aneffective amount of the composition of claim
 24. 29. A method oftreating cancer in a subject, comprising administering to the subject aneffective amount of an antibody or antigen-binding fragment thereof thatcompetes with an antibody according to anyone of claims 1 to 18 for thebinding of CD38.
 30. The method of claim 28 or 29, further comprisingadministering, simultaneously or sequentially in any order, a secondagent to the subject.
 31. The method according to anyone of claim 28, 29or 30 wherein the subject has a solid tumor.
 32. The method according toanyone of claim 28, 29 or 30 wherein the subject has a haematologicalcancer.
 33. A method of preparing an anti-CD38 antibody comprisingproviding an antibody according to any one of claims 1 to 18, andsubjecting the antibody to affinity maturation, wherein the anti-CD38antibody produced has a greater affinity to CD38 than the parentalantibody.
 34. A method of preparing a pharmaceutical compositioncomprising providing an antibody prepared according to a method of claim33 and co-formulating the antibody with at least one or morepharmaceutically acceptable excipients.
 35. A kit comprising thecomposition of claim 23 in a container.